Lab Ultrafiltration

and Purification
Products
Table of Contents

General Information
Ultrafiltration Applications 4
Lab Ultrafiltration Devices 5
Ultrafiltration Process Methods 5
Membrane Performance Characteristics 6
Membrane Selection Guide 7

---
Protein and Macromolecule Concentration 9
Centrifugal and Pressurized Ultrafiltration

---
Vivaspin® 500 10
Vivaspin® 2 12
Vivaspin® Filtrate 14

---
Vivaspin® Turbo 4 PES 16
Vivaspin® 6 18
Vivaspin® 15R 20

---
Vivaspin® Endotest 22
Vivaspin® Turbo 15 PES 24
Vivaspin® Turbo 15 RC 26
Vivaspin® 20 28
Vivaspin® 100 30

---
Vivaspin® Equipment and Accessories 32

Tangential Flow Filtration

-
Vivaflow® 50 34
Vivaflow® 50R 36
Vivaflow® 200 38
Vivaflow® Equipment and Accessories 40

Ultrafiltration Membrane Discs 42

2
DNA Concentration 45
Vivacon® 500 46
Vivacon® 2 49

Protein Purification 53
Vivaclear Centrifugal Filters 54
Vivapure® Ion Exchange Purification Products 56

Virus Purification and Concentration 59

---
Vivapure® Virus Purification and Concentration Kits 60

Adenovirus Purification 61
Vivapure® Adenopack 20 62
Vivapure® Adenopack 100 63

---
5Vivapure® Adenopack 500 65

Lentivirus Purification 66
Vivapure® Lentiselect 40 67
Vivapure® Lentiselect 500 68
Vivapure® Lentiselect 1000 69

Application Notes 71
1. Desalting and Buffer Exchange with Vivaspin® Centrifugal
Concentrators 72
2. Treatment of Vivaspin® Concentrators for Improved Recovery
of Low-Concentrated Protein Samples 75
3. Scouting Protein Purification Conditions Using Vivapure® Centrifugal
Ion Exchange Membrane Absorbers 78
4. Concentration and Purification of Viruses by using Ultrafiltration,
Incl. Coronavirus - a Short Review 83
5. Sartorius Ultrafiltration Products in the Preparation of Biological
Nanoparticles and Medical Nanocarriers 95
6. Vivaflow® and Vivaspin® Workflow in Protein Research Laboratories 101

3
 Ultrafiltration Applications

Ultrafiltration is a convective process Solute Concentration

using anisotropic semi-permeable Ultrafiltration membranes are used
membranes to separate to increase the solute concentration
macromolecular species and solvents of a desired biological or inorganic
– primarily on the basis of size. It is species. Macromolecules are retained
particularly appropriate for the by the membrane when they are sig-
concentration of macromolecules nificantly larger than the nominal pore
and can also be used for purification size, while salts and microsolutes are
or solvent ­exchange. Ultrafiltration is removed with the solvent.
Solute concentration a gentle, non-denaturing method
that is more efficient and flexible Solute Fractionation

-
than ­alternative processes. Ultrafiltration is a cost effective
method for separating samples into
Typical Applications size-graded components providing
Concentration | desalting of proteins, that the desired fractions have at

--
enzymes, DNA, monoclonal antibod- least a 10-fold difference in molecular
ies, immunoglobulins, extracellular weight. During filtration, the
vesicles, viruses and nanoparticles permeating solute remains at its initial
Forensic DNA sample concentration ­concentration whilst the retained

--
prior to sequencing reaction ­macromolecules will be enriched.
Solute fractionation Peptide fractionation in FASP
(filter-aided sample preparation) Solute Desalting or Purification

--
Free drug | hormone assays A solution may be purified from salts,
Removal of primers from PCR solvents and low molecular weight
amplified DNA materials by diafiltration. Multiple

--
Removal of labeled amino acids solvent exchanges will progressively
and nucleotides purify macromolecules from
HPLC sample preparation contaminating microsolutes, which are

--
Deproteinization of samples typically removed most efficiently by
Recovery of biomolecules from cell adding solvent to the sample at a rate
culture supernatants | lysates equal to the speed of filtration. This is

-
Solute desalting or purification Mammalian cell harvesting called continuous diafiltration, and it
Cell washing, virus purification, cell replaces time-intensive techniques
debris removal and depyrogenation such as dialysis.
Environmental sample clarification |
concentration

4 General Information
 Lab Ultrafiltration Devices

Sartorius develops devices dedicated support tools to help users select the
to optimizing laboratory ultrafiltration optimum device and process for their
processes with minimal time sample type.
requirements while maximizing
Vivaspin® Turbo

1.5

recovery, reliability and robustness. Visit www.sartorius.com for more

1.0

0.5

technical and application support

In addition Sartorius are continually material.
building technical and application

Centrifugal

Ultrafiltration Process Methods

Sartorius offers a comprehensive Pressure-Fugation

choice of operating methods for (5 to 15 mL starting volumes)
ultrafiltration and diafiltration. The A unique Sartorius method that
guidance below will support you in combines gas pressure with
selecting the most suitable method, centrifugation, with process times
Pressure and pressure-fugation depending on sample volume, typically 30 to 50% faster than
­equipment available, and the desired centrifugation alone. Vivaspin® 20 can
filtration speed and process control. be operated this way.

Centrifugal Crossflow | TFF

(0.1 to 90 mL starting volumes) (0.1 to 5 L starting volumes)
Driven by centrifugal force, solvent and The solution to be processed is pumped
microsolutes are cleared through the under pressure across an ultrafiltration
ultrafiltration membrane and into a membrane and then returned to the
filtrate container, usually positioned original reservoir. The solution is
below. This gentle process is quick to progressively concentrated or purified
Crossflow | TFF set up and offers fast filtration speeds as solvent and microsolutes pass
with most solutions. Twelve centrifugal through the membrane into a separate
devices are offered from the Vivaspin® filtrate vessel. Vivaflow® cassettes are
and Vivacon® families. offered for this method.

Pressure
(5 to 98 mL starting volumes)
Pressurized air or an inert gas provide
the vector for ultrafiltration. For
increased process speed, pressurized
devices can be placed on an orbital
shaker, where agitation impedes
macromolecules from polarizing on
the membrane surface. Vivaspin® 20
and 100 can be operated using gas
pressure.

General Information 5
 Membrane Performance Characteristics

Sartorius offers an extended range of This is a hydrophillic membrane

membranes to cover the majority of suitable for general samples, with
ultrafiltration requirements. The ultra-low protein adsorption and high
following is a guide to selecting the chemical compatibility.
most appropriate membranes Regenerated cellulose is especially
according to their typical well suited to ultrafiltration of
performance characteristics. oligonucleotides and peptides.
However, membrane behavior and
performance can be highly Hydrosart® (HY)
dependent on the specific Demonstrating the same properties as
characteristics of each sample. regenerated cellulose, but with the
Therefore, it is recommended to added benefit of enhanced
experiment with multiple membrane performance characteristics and
materials when optimizing your extremely low protein binding.
ultrafiltration process. Hydrosart® is another membrane of
choice for applications such as
Polyethersulfone (PES) concentration and desalting of
This is a low binding membrane that immunoglobulin fractions.
provides excellent performance with
most solutions and exceptional Cellulose Triacetate (CTA)
recovery of negatively charged target High hydrophilicity and very low non-
molecules. Polyethersulfone specific binding characterize this
membranes are usually preferred for membrane. Cast without any support
their low fouling characteristics, that could trap or bind passing
exceptional flux and broad pH microsolutes, these membranes are
compatibility. preferred for sample cleaning and
protein removal, and when high
Regenerated Cellulose (RC) recoveries from the filtrate solution is
The Sartorius regenerated cellulose of primary importance.
membrane has been uniquely
developed to ensure optimal
performance in the lab ultrafiltration
devices.

Membrane Performance Comparisons

Membrane Frequently preferred for:

Polyethersulfone & Regenerated Cellulose Concentration

3 kDa MWCO Desalting
5 kDa MWCO Buffer exchange
10 kDa MWCO Fractionation
30 kDa MWCO
50 kDa MWCO
100 kDa MWCO

Cellulose triacetate Deproteinization

5 kDa MWCO Free|bound drug studies
10 kDa MWCO Whenever the filtrate is being analyzed
20 kDa MWCO

Hydrosart® Concentration
2 kDa MWCO Desalting
5 kDa MWCO Buffer exchange
10 kDa MWCO Fractionation
30 kDa MWCO Membrane evaluation for scale up

6 General Information
Membrane Selection Guide

The advanced designs and low surface of the sample container. Whilst
­adsorption materials that characterize the relative adsorption will be
­Sartorius ultrafilters, offer a unique proportionately less important than on
­combination of faster processing the membrane, due to the higher total
speeds and highest target molecule surface area, this can be the major
recoveries. Providing that the source of yield loss.
appropriate sample capacity,
membrane material and MWCO are Process Optimization
selected, these devices will ­typically When the highest recoveries are
yield recoveries in excess of 90% when crucial, particularly when working with

--
the initial sample contains > 0.1 mg/mL solute quantities in the microgram
of the solute of interest. The majority of range, Sartorius recommends that
any loss occurs through non-­specific users consider the following:
binding to the membrane surface and | Select the smallest device that suits
or the sample container polymer. the sample volume.

--
Take advantage of the extra speed
Adsorption to the Membrane of Sartorius products by refilling a
Depending on sample characteristics smaller device repeatedly.
relative to the membrane type used, Select the lowest MWCO membrane
solute adsorption on the membrane that suits the application.

-
surface is typically 2-10 μg/cm2. This can Reduce pressure or centrifugal
increase to 20-100 μg/cm2 when the force to approximately half of the
­filtrate is of interest and the solute must recommended maximum.
pass through the whole internal Avoid over-concentration. The

-
structure of the membrane. Typically a smaller the final concentrate volume,
higher cut-off membrane will bind the more difficult it is to achieve
more than a low molecular weight complete recovery.

-
alternative. If feasible, after sample retrieval,
rinse the device with one or more
Adsorption to the Sample Container drops of buffer.
Although every effort is made to Pretreat the device overnight with a
minimize this phenomenon by the passivation solution such as 5% SDS,
selection of low binding materials and Tween 20 or Triton X-100, then rinse
tool production to optical standards, thoroughly before use.
some solute will bind to the internal

Membrane Selection Guide (Recommended MWCO)

Application < 5 kDa 10 kDa 30 kDa 50 kDa 100 kDa > 300 kDa
Bacteria
Enzymes
Extracellular vesicles
Growth factors
IgG and mAbs
Nucleic acids
Oligonucleotides
Peptides
Viruses
Yeast

For highest recovery, select a membrane MWCO which is a maximum of one third to half the
molecular weight of the solute to be retained

General Information 7
8 Chapter Site Title
Protein and Macromolecule
Concentration
Table of Contents

Vivaspin® 500 10

Vivaspin® 2 12

Vivaspin® Filtrate 14

Vivaspin® Turbo 4 PES 16

Vivaspin® 6 18

Vivaspin® 15R 20

Vivaspin® Endotest 22

Vivaspin® Turbo 15 PES 24

Vivaspin® Turbo 15 RC 26

Vivaspin® 20 28

Vivaspin® 100 30

Vivaspin® Equipment and Accessories 32

Vivaflow® 50 34

Vivaflow® 50R 36

Vivaflow® 200 38

Vivaflow® Equipment and Accessories 40

Ultrafiltration Membrane Discs 42

9
 Vivaspin® 500

100 to 500 μL samples The legacy patented vertical

Vivaspin® 500 centrifugal filter units membrane design and thin channel
offer a simple, one step procedure for filtration chamber (US 5,647,990),
sample preparation. They can minimize membrane fouling and
effectively be used in fixed angle provide fast concentrations – even
rotors a
­ ccepting 2.2 mL centrifuge with particle-loaded s­ olutions.
tubes.

Technical Specifications

Concentrator capacity

Swing bucket rotor do not use

Fixed angle rotor 500 µL

Dimensions
100

50 75
Length x diameter 50 x 11 mm
50

Active membrane area 0.5 cm²

Hold-up volume, membrane and support < 5 µL

100
75

50

Dead-stop volume 5 µL

Materials of construction

11 Body Polycarbonate (PC)

Filtrate vessel Polypropylene (PP)

Concentrator cap Polycarbonate (PC)

Membrane Polyethersulfone (PES)

Equipment Required

Centrifuge

Rotor type Fixed angle (min. 40°)

Rotor cavity To fit 2.2 mL (11 mm) conical bottom tubes

Maximum RCF 12,000 g

Concentrate recovery

Pipette type Fixed or variable volume

Recommended tip Thin gel loader type

10 Protein Concentration Centrifugal Filtration Vivaspin® 500

Typical Performance Characteristics

Time to concentrate up to 30x

at 20°C and solute recovery

Rotor Fixed angle

Centrifugal force 12,000 g

Start volume 500 µL

Time Recovery

Aprotinin 0.25 mg/mL (6.5 kDa)

3 kDa MWCO PES 30 min 96%

BSA 1.0 mg/mL (66 kDa)

5 kDa MWCO PES 15 min 96%
10 kDa MWCO PES 5 min 96%
30 kDa MWCO PES 5 min 95%

IgG 0.25 mg/mL (160 kDa)

30 kDa MWCO PES 10 min 96%
50 kDa MWCO PES 10 min 96%
100 kDa MWCO PES 10 min 96%

Ordering Information

Vivaspin® 500 PES 25 pc 100 pc

3 kDa MWCO VS0191 VS0192

5 kDa MWCO VS0111 VS0112

10 kDa MWCO VS0101 VS0102

30 kDa MWCO VS0121 VS0122

50 kDa MWCO VS0131 VS0132

100 kDa MWCO VS0141 VS0142

300 kDa MWCO VS0151 VS0152

1,000 kDa MWCO VS0161 VS0162

0.2 µm VS0171 VS0172

Protein Concentration Centrifugal Filtration Vivaspin® 500 11

 Vivaspin® 2

0.4 to 3 mL samples Also unique to Vivaspin® 2 is the

Vivaspin® 2 bridges the gap between choice of directly pipetting the
the 500 μL and 4 mL centrifugal concentrate from the dead-stop
concentrators. This device combines pocket built into the bottom of the
the speed of the classic Vivaspin® concen­trator, or alternatively reverse
products with low internal surface and spinning into the concentrator
membrane areas for superior recovery cap. Both methods result in
recoveries from very dilute solutions. near total concentrate recoveries.

Available with a choice of

polyethersulfone, Hydrosart® or
cellulose triacetate membranes,
Vivaspin® 2 offers the highest flexibility
for process optimization.
2.0
mL
1.5

1.5

Technical Specifications
1.0
100K
1.0
MWCO

126
200
µL
Concentrator capacity
100

20 Swing bucket rotor 3 mL

Fixed angle rotor 2 mL

Dimensions

Length x diameter 126 x 17 mm

Active membrane area 1.2 cm2

17
Hold-up volume, membrane < 10 μL

Dead-stop volume 8 μL

Materials of construction

Body Polycarbonate (PC)

Filtrate vessel Polycarbonate (PC)

Concentrator cap Polycarbonate (PC)

Membrane Polyethersulfone (PES)

Hydrosart® (HY)
Cellulose Triacetate (CTA)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 15 mL (17 mm) To fit 15 mL (17 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 4,000 g 8,000 g

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

12 Protein Concentration Centrifugal Filtration Vivaspin® 2

 Typical Performance Characteristics

2.0
mL
1.5
Time to concentrate up to 30×
at 20°C and solute recovery
1.5

1.0
100K
1.0
MWCO

Rotor Fixed angle

Integral deadstop 200
µL
100

avoids risk of 20

Centrifugal force 5,000 g

concentrating
Start volume 2 mL
to dryness
Time Recovery

PES, HY or CTA membranes Insulin chain A 0.1 mg/mL (2.5 kDa)

2 kDa MWCO HY 35 min 95%

Aprotinin 0.25 mg/mL (6.5 kDa)

3 kDa MWCO PES 50 min 96%
20

100
BSA 1.0 mg/mL (66 kDa)
5 kDa MWCO PES 12 min 98%
µL
200

MWCO
1.0
5 kDa MWCO HY 22 min 98%
10 kDa MWCO PES 8 min 98%
100K
1.0

1.5

10 kDa MWCO CTA 10 min 96%

1.5
mL
2.0

10 kDa MWCO HY 12 min 98%

20 kDa MWCO CTA 5 min 96%
30 kDa MWCO PES 8 min 97%
30 kDa MWCO HY 5 min 97%

IgG 0.25 mg/mL (160 kDa)

20 kDa MWCO CTA 6 min 97%
Reverse spin concentrate retrieval 30 kDa MWCO PES 10 min 96%
50 kDa MWCO PES 10 min 96%
100 kDa MWCO PES 8 min 95%

Ordering Information

Vivaspin® 2 PES 25 pc 100 pc

3 kDa MWCO VS0291 VS0292

5 kDa MWCO VS0211 VS0212

10 kDa MWCO VS0201 VS0202

30 kDa MWCO VS0221 VS0222

50 kDa MWCO VS0231 VS0232

100 kDa MWCO VS0241 VS0242

300 kDa MWCO VS0251 VS0252

1,000 kDa MWCO VS0261 VS0262

0.2 μm VS0271 VS0272

Vivaspin 2 CTA
®

10 kDa MWCO VS02V1 VS02V2

20 kDa MWCO VS02X1 VS02X2

Vivaspin 2 HY
®

2 kDa MWCO VS02H91 VS02H92

5 kDa MWCO VS02H11 VS02H12

10 kDa MWCO VS02H01 VS02H02

30 kDa MWCO VS02H21 VS02H22

Protein Concentration Centrifugal Filtration Vivaspin® 2 13

 Vivaspin® Filtrate

0.5–2.5 mL samples The ultrafiltrate is collected in the

Vivaspin Filtratre® is a ready-to-use floating filtrate tube, where it is readily
unit for low volume centrifugal accessible without disassembly.

---
ultrafiltration to separate proteins from
low molecular weight substances in Vivaspin® Filtrate is ideal for the
biological samples. following applications:

--
Drug binding studies
Vivaspin Filtratre® features a unique Isolation of metabolites from serum
design that enables ultrafiltration in Protein removal from blood samples
the direction opposite to centrifugal Cleaning of liposomes
force. This is so effective in preventing Virus removal
premature blockage of the filter that
even whole blood samples can be
deproteinized.

Technical Specifications

Concentrator capacity
Swing bucket rotor 2.5 mL
93
Fixed angle rotor 2.5 mL
Dimensions
Length x diameter 93 x 14 mm
Active membrane area 0.79 cm2
Hold-up volume, membrane < 5 μL
max. 2.5 mL

Dead-stop volume 100 μL

Materials of construction
14
Centrifuge tube Polystyrene (PS)
Filtrate tube Styrene Acrylonitrile (SAN)
Concentrator cap Polyethylene (PE)
Membrane Cellulose Triacetate (CTA)
Polyethersulfone (PES)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 15 mL (17 mm) To fit 15 mL (17 mm)

conical | flat bottom tubes conical | flat bottom tubes

Maximum RCF 2,500 g 2,000 g

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

14 Protein Concentration Centrifugal Filtration Vivaspin® Filtrate

Easy-to-use Typical Performance Characteristics

Time to filter Time to filter Passage of

50% of sample 90% of sample sample species
volume volume volume

Centrifugal force 2,000 g

Start volume 2.5 mL

BSA 1.0 mg/mL (66 kDa)

5 kDa MWCO 300 min – 0%
10 kDa MWCO 35 min 80 min 2%
20 kDa MWCO 9 min 20 min 2%
Remove filtrate tube, pour in sample Blue Dextran 0.1 mg/mL (2,000 kDa)
300 kDa MWCO 9 min 25 min 28%

Ordering Information

Vivaspin® Filtrate CTA 12 pc

5 kDa MWCO 13229-E

10 kDa MWCO 13239-E

20 kDa MWCO 13249-E

Replace filtrate tube Vivaspin Filtrate PES

®

300 kDa MWCO 13279-E

References R. H. Christenson, S. D. Studenberg,

P. Nebinger and Koel (1993). S. Beck-Davis and F. A. Sedor (1987).
Determination of acyclovir by Digoxin-like immunoreactivity
Centrifuge ultrafiltration and high-performance eliminated from serum by centrifugal
liquid chromatography. ultrafiltration before fluorescence
J. Chromatography 619, 342-344 polarization immunoassay of digoxin.
Clinical Chemistry 33, 606-608
F. da Fonseca-Wollheim, K.-G. Heinze,
K. Lomsky and H. Schreiner (1988).
Serum ultrafiltration for the elimination
of endogenous interfering substances
in creatinine determination.
J. Clin. Chem. Clin. Biochem. 26,
523-525
Recover …or use forceps
the filtrate… to remove the
filtrate tube
and access the
concentrate

Protein Concentration Centrifugal Filtration Vivaspin® Filtrate 15

 Vivaspin® Turbo 4 PES

2 to 4 mL samples The optimized design and sleek

Vivaspin® Turbo 4 PES offers the internal profile ensure maximum
fastest sample concentration with the process speeds all the way down to the
highest recoveries. This device can last few micro­liters, resulting in more
handle up to 4 mL sample volumes in than ­100-fold concentration.
swing bucket and fixed angle rotors
that accept 15 mL conical bottom UV joining technology provides a
­centrifuge tubes. smooth transition between membrane
and housing, allowing collection of the
entire concentrated sample from the
unique, pipette tip-friendly angular
dead-stop pocket.

Technical Specifications

Concentrator capacity
122
Swing bucket rotor 4 mL
Fixed angle rotor 4 mL
Dimensions
Length x diameter 122.5 x 17 mm
Active membrane area 3.2 cm2
Hold-up volume, membrane < 10 μL

17 Dead-stop volume, swing bucket 40 μL

Dead-stop volume, fixed angle 30 μL

Materials of construction
Body Styrene Butadiene Copolymer (SBC)
Filtrate vessel Polypropylene (PP)
Concentrator cap Polypropylene (PP)
Membrane Polyethersulfone (PES)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 15 mL (17 mm) To fit 15 mL (17 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 4,000 g 7,500 g

Maximum RCF, 100 kDa MWCO 3,000 g 5,000 g

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

16 Protein Concentration Centrifugal Filtration Vivaspin® Turbo 4

Visit us at Typical Performance Characteristics
www.sartorius.com/
VivaspinTurbo4 Time to concentrate up to 30×
for further information. at 20°C and solute recovery

--
Here you can find instruc- Rotor Swing bucket Fixed angle (25°)
tions on how to use Vivaspin® Centrifugal force* 4,000 g 7,500 g
Turbo 4 PES for:
Start volume 4 mL 4 mL
Desalting and buffer
Time Recovery Time Recovery
exchange

--
Preparation of biological Cytochrome c (12.4 kDa)
nanoparticles and medical 3 kDa MWCO PES 60 min 98% 80 min 96%
5 kDa MWCO PES 40 min 95% 50 min 94%
nanocarriers
Concentration and Lysozyme (14.3 kDa)
3 kDa MWCO PES 65 min 95% 70 min 93%

-
purification of viruses 5 kDa MWCO PES 50 min 94% 60 min 92%
Urine protein concentra-
α-Chymotrypsin (25 kDa)
tion
10 kDa MWCO PES 10 min 95% 8 min 95%
Separation of proteins
BSA (66 kDa)
and metabolites for disease
10 kDa MWCO PES 10 min 98% 7 min 97%
detection 30 kDa MWCO PES 8 min 96% 6 min 97%

IgG (160 kDa)

30 kDa MWCO PES 18 min 94% 13 min 92%
50 kDa MWCO PES 16 min 93% 12 min 90%
100 kDa MWCO PES 17 min 94% 13 min 92%

Ordering Information

Vivaspin® Turbo 4 PES 25 pc 100 pc

3 kDa MWCO VS04T91 VS04T92

5 kDa MWCO VS04T11 VS04T12

10 kDa MWCO VS04T01 VS04T02

30 kDa MWCO VS04T21 VS04T22

50 kDa MWCO VS04T31 VS04T32

100 kDa MWCO VS04T41 VS04T42

* 3,000 g (swing bucket) or 5,000 g (fixed

angle) centrifugal force for 100 kDa MWCO
devices.

Protein Concentration Centrifugal Filtration Vivaspin® Turbo 4 17

 Vivaspin® 6

2 to 6 mL samples Featuring twin vertical membranes for

Vivaspin® 6 concentrators have been unparalleled filtration speeds and
developed to offer increased more than 100-fold ­concentration, the
volume flexibility and performance. retentate volume is easily estimated
from the printed graduations on the
Vivaspin® 6 can process an impressive side of the concentrator. The modified
6 mL in either swing bucket or fixed dead-stop pocket further simplifies
angle rotors accepting standard 15 mL direct pipette ­retrieval of the final
conical bottom centrifuge tubes. concentrate.

Technical Specifications
5

3 Concentrator capacity
2
Swing bucket rotor 6 mL
1

0.5
Fixed angle rotor 6 mL
0.3

Dimensions
0.2

122 0.1

Length x diameter 122 x 17 mm

Active membrane area 2.5 cm2
Hold-up volume, membrane < 10 μL
Dead-stop volume 30 μL

Materials of construction
Body Polycarbonate (PC)
17
Filtrate vessel Polycarbonate (PC)
Concentrator cap Polypropylene (PP)
Membrane Polyethersulfone (PES)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 15 mL (17 mm) To fit 15 mL (17 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 4,000 g 8,000 g

Maximum RCF, 4,000 g 6,000 g

≥100 kDa MWCO

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

18 Protein Concentration Centrifugal Filtration Vivaspin® 6

Performance Characteristics

Time to concentrate up to 30×

at 20°C and solute recovery

Rotor Swing bucket Fixed angle (25°)

Centrifugal force 3,000 g 7,500 g

Start volume 6 mL 6 mL

Time Recovery Time Recovery

Cytochrome c 0.25 mg/mL (12.4 kDa)

3 kDa MWCO PES – – 90 min 97%

BSA 1.0 mg/mL (66 kDa)

5 kDa MWCO PES 20 min 98% 12 min 98%
10 kDa MWCO PES 13 min 98% 10 min 98%
30 kDa MWCO PES 12 min 98% 9 min 97%

IgG 0.25 mg/mL (160 kDa)

30 kDa MWCO PES 18 min 96% 15 min 95%
50 kDa MWCO PES 17 min 96% 14 min 95%
100 kDa MWCO PES 15 min 91% 12 min 91%

Latex beads 0.004% in DMEM + 10% FCS

(55 nm)
300 kDa MWCO PES – – 25 min 99%

Latex beads 0.004% in DMEM + 10% FCS

(240 nm)
1,000 kDa MWCO PES – – 4 min 99%

Yeast 1.0 mg/mL (S. Cerevisiae)

0.2 μm PES 4 min 97% 3 min 97%

Ordering Information

Vivaspin® 6 PES 25 pc 100 pc

3 kDa MWCO VS0691 VS0692

5 kDa MWCO VS0611 VS0612

10 kDa MWCO VS0601 VS0602

30 kDa MWCO VS0621 VS0622

50 kDa MWCO VS0631 VS0632

100 kDa MWCO VS0641 VS0642

300 kDa MWCO VS0651 VS0652

1,000 kDa MWCO VS0661 VS0662

0.2 μm VS0671 VS0672

Protein Concentration Centrifugal Filtration Vivaspin® 6 19

 Vivaspin® 15R

--
--
2 to 15 mL samples Ultimate recoveries (95 – 98%)
Vivaspin® 15R is designed for initial Extremely short concentration time
sample volumes up to 15 mL and (30-fold in 15 minutes)
­features a modified regenerated Simple and convenient handling

-
­cellulose membrane; Hydrosart®. Easy scale-up to 0.1 to 5 L with
This membrane is ideal where ­Vivaflow® 50R or 200 with
extremely high recovery with very low ­Hydrosart® ­membranes
adsorption is needed. An example of Very low hold-up volume (< 20 μL)
this application includes desalting and
concentration of immunoglobulin
fractions.

15

10
Technical Specifications
5

Concentrator capacity
1.0

116 Swing bucket rotor 15 mL

0.75

0.5

0.3

0.15

Fixed angle rotor 12.5 mL

Dimensions

Length x diameter 116 x 30 mm

Active membrane area 3.9 cm2

Hold-up volume, membrane < 20 μL

30
Dead-stop volume 30 μL

Materials of construction

Body Polycarbonate (PC)

Filtrate vessel Polycarbonate (PC)

Concentrator cap Polypropylene (PP)

Membrane Hydrosart® (HY)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 50 mL (30 mm) To fit 50 mL (30 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 3,000 g 6,000 g

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

20 Protein Concentration Centrifugal Filtration Vivaspin® 15R

 Typical Performance Characteristics
0.15

0.3

0.5

0.75
1.0

Time to concentrate up to 30×

5

10

at 20°C and solute recovery

15

Rotor Swing bucket Fixed angle (25°)

0.75
0.15

0.5
0.3

1.0

10

10
15

15
5

0.75

0.15
0.5

0.3
1.0

Centrifugal force 3,000 g 6,000 g

15

10

1.0

Start volume 15 mL 12.5 mL

0.75

0.5

0.3

0.15

Time Recovery Time Recovery

Spin Aprotinin 0.1 mg/mL* (6.5 kDa)

5 kDa MWCO 47 min 95% 45 min 95%

Cytochrome c 0.25 mg/mL* (12.4 kDa)

5 kDa MWCO 45 min 96% 45 min 96%
10 kDa MWCO 25 min 94% 18 min 94%

15
α-chymotrypsin 0.25 mg/mL* (25 kDa)
10 5 kDa MWCO 50 min 98% 45 min 98%
10 kDa MWCO 25 min 98% 18 min 98%
5

1.0
0.75

0.5

Ovalbumin 1.0 mg/mL* (45 kDa)

0.3

0.15

10 kDa MWCO 20 min 98% 14 min 98%

30 kDa MWCO 15 min 94% 12 min 94%

BSA 1.0 mg/mL* (66 kDa)

Recover 30 kDa MWCO 18 min 98% 15 min 98%

IgG 0.1 mg/mL in DMEM (160 kDa)

30 kDa MWCO 30 min 98% 25 min 96%

Ordering Information

Vivaspin® 15R HY 12 pc 48 pc

2 kDa MWCO VS15RH91 VS15RH92

5 kDa MWCO VS15RH11 VS15RH12

10 kDa MWCO VS15RH01 VS15RH02

30 kDa MWCO VS15RH21 VS15RH22

* Proteins other than IgG made up in 50 mM

potassium phosphate, 150 mM sodium
chloride, pH 7.4

Protein Concentration Centrifugal Filtration Vivaspin® 15R 21

 Vivaspin® Endotest

2 to 15 mL samples The centrifugal design enables parallel

Vivaspin® Endotest is a single-use preparation of multiple test samples,
ultrafiltration device for endotoxin minimizing hands-on time.
concentration and removal of
interfering substan ces from liquid Vivaspin® Endotest can effectively be
samples prior to LAL testing. used in swing bucket or fixed angle
rotors accepting 50 mL conical
These devices are certified pyrogen bottom centrifuge tubes.
free (≤ 0.05 EU/mL) and are available
with 20 kDa MWCO cellulose
triacetate membranes.

15

10
Technical Specifications
5

Concentrator capacity
1.0

116 Swing bucket rotor 15 mL

0.75

0.5

0.3

0.15

Fixed angle rotor 12.5 mL

Dimensions

Length x diameter 116 x 30 mm

Active membrane area 3.9 cm2

Hold-up volume, membrane < 20 μL

30
Dead-stop volume 30 μL

Materials of construction

Body Polycarbonate (PC)

Filtrate vessel Polycarbonate (PC)

Concentrator cap Polypropylene (PP)

Membrane Cellulose Triacetate (CTA)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 50 mL (30 mm) To fit 50 mL (30 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 1,000 g 1,000 g

Sample Mixing

Laboratory mixer Vortex

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

22 Protein Concentration Centrifugal Filtration Vivaspin® Endotest

 Ordering Information
0.15

0.3

0.5

0.75
1.0

Vivaspin® Endotest CTA 12 pc

5

10

15

20 kDa MWCO VS15RXETO

0.75
0.15

0.5
0.3

1.0

10

10
15

15
5

0.75

0.15
0.5

0.3
1.0

15

10

1.0
0.75

0.5

0.3

0.15

Spin

15

10

1.0
0.75

0.5

0.3

0.15

Recover

Protein Concentration Centrifugal Filtration Vivaspin® Endotest 23

 Vivaspin® Turbo 15 PES

4 to 15 mL samples UV joining technology provides a

Vivaspin® Turbo 15 PES enables the smooth transition between membrane
fastest sample concentration and and housing, allowing collection of the
highest recoveries. This device can entire concentrated sample from the
handle samples up to 15 mL in rotors unique, angular dead-stop pocket.
­accepting 50 mL c ­ entrifuge tubes.
The optimized design and sleek inter- Stable polyethersulfone membranes
nal profile ensure maximum process are suited to a wide pH range and espe-
speeds all the way down to the last few cially recommended for high recovery
microlitres, resulting in more than of negatively charged target molecules.
­100-fold concentration. Now complemented with a regenerat-
ed cellulose option, Vivaspin® Turbo
offers the best membrane, whatever
the sample.
Vivaspin® Turbo

1.5

118 1.0 Technical Specifications

0.5

Concentrator capacity

Swing bucket rotor 15 mL

Fixed angle rotor 11 mL

Dimensions

30 Length x diameter 118 x 30 mm

Active membrane area 7.2 cm2

Hold-up volume, membrane <10 μL

Dead-stop volume, swing bucket | fixed angle 100 | 60 μL

Materials of construction

Body Styrene Butadiene Copolymer (SBC)

Filtrate vessel Polypropylene (PP)

Concentrator cap Polypropylene (PP)

Membrane Polyethersulfone (PES)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 15 mL (17 mm) To fit 15 mL (17 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 4,000 g 4,000 g

Maximum RCF, 2,000 g 2,000 g

100 kDa MWCO

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

24 Protein Concentration Centrifugal Filtration Vivaspin® Turbo 15 PES

Typical Performance Characteristics

Time to concentrate up to 20×

at 20°C and solute recovery

Rotor Swing bucket Fixed angle (25°)

Centrifugal force 4,000 g 4,000 g

Start volume 15 mL 11 mL

Time Recovery Time Recovery

Cytochrome c 0.25 mg/mL (12.4 kDa)

5 kDa MWCO PES 30 min 98% 50 min 98%

Lysozyme 0.25 mg/mL (14.3 kDa)

5 kDa MWCO PES 33 min 96% 50 min 96%

α-Chymotrypsin 1.0 mg/mL (25 kDa)

10 kDa MWCO PES 10 min 95% 10 min 95%

BSA 1.0 mg/mL (66 kDa)

10 kDa MWCO PES 10 min 99% 10 min 99%
30 kDa MWCO PES 8 min 98% 10 min 98%

IgG 1.0 mg/mL (160 kDa)

30 kDa MWCO PES 23 min 95% 17 min 95%

Ordering Information

Vivaspin® Turbo 15 PES 12 pc 48 pc

3 kDa MWCO VS15T91 VS15T92

5 kDa MWCO VS15T11 VS15T12

10 kDa MWCO VS15T01 VS15T02

30 kDa MWCO VS15T21 VS15T22

50 kDa MWCO VS15T31 VS15T32

100 kDa MWCO VS15T41 VS15T42

Protein Concentration Centrifugal Filtration Vivaspin® Turbo 15 PES 25

 Vivaspin® Turbo 15 RC

4 to 15 mL samples complete concentrate ­recovery from

Vivaspin® Turbo 15 RC enables the the unique, a
­ ngular dead-stop pocket.
fastest sample concentration and
highest recoveries. This device can Regenerated cellulose membranes
handle samples up to 15 mL in rotors developed specifically for Sartorius
­accepting 50 mL c ­ entrifuge tubes. lab ultrafiltration devices are suited
The optimized design and sleek to general samples, with ultra-low
internal profile ensure maximum ­adsorption and high chemical
process speeds all the way down to the compatibility, and especially
last few microlitres, resulting in more recommended for oligonucleotides
than 100-fold concentration. and peptides. ­Complemented with a
polyethersulfone option, ­Vivaspin®
Solvent-free, heat weld technology Turbo offers the best ­membrane,
provides a smooth transition between whatever the sample.
membrane and housing, ­allowing
Vivaspin® Turbo

1.5

118 1.0 Technical Specifications

0.5

Concentrator capacity

Swing bucket rotor 15 mL

Fixed angle rotor 11 mL

Dimensions

30 Length x diameter 118 x 30 mm

Active membrane area 8.1 cm2

Hold-up volume, membrane < 28 μL

Dead-stop volume, swing bucket | fixed angle 120 | 140 μL

Materials of construction

Body Styrene Butadiene Copolymer (SBC)

Filtrate vessel Polypropylene (PP)

Concentrator cap Polypropylene (PP)

Membrane Regenerated Cellulose (RC)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 15 mL (17 mm) To fit 15 mL (17 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 4,000 g 6,000 g

Maximum RCF, 3,000 g 6,000 g

100 kDa MWCO

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

26 Protein Concentration Centrifugal Filtration Vivaspin® Turbo 15 RC

 Typical Performance Characteristics

Time to concentrate up to 20×

at 20°C and solute recovery

Rotor Swing bucket Fixed angle (25°)

Centrifugal force 4,000 g* 6,000 g

Start volume 15 mL 11 mL

Time Recovery Time Recovery

Cytochrome c 0.25 mg/mL (12.4 kDa)

5 kDa MWCO RC 23 min 94% 37 min 92%

Lysozyme 0.25 mg/mL (14.3 kDa)

5 kDa MWCO RC 23 min 94% 37 min 89%

α-Chymotrypsin 1.0 mg/mL (25 kDa)

10 kDa MWCO RC 7 min 93% 9 min 92%

BSA 1.0 mg/mL (66 kDa)

10 kDa MWCO RC 8 min 94% 10 min 98%
30 kDa MWCO RC 4 min 96% 4 min 93%

IgG 1.0 mg/mL (160 kDa)

50 kDa MWCO RC 17 min 95% 11 min 96%
100 kDa MWCO RC 18 min 89% 12 min 89%

Ordering Information

Vivaspin® Turbo 15 RC 12 pc 48 pc

5 kDa MWCO VS15TR11 VS15TR12

10 kDa MWCO VS15TR01 VS15TR02

30 kDa MWCO VS15TR21 VS15TR22

50 kDa MWCO VS15TR31 VS15TR32

100 kDa MWCO VS15TR41 VS15TR42

* 3,000 g for 100 kDa MWCO devices

Protein Concentration Centrifugal Filtration Vivaspin® Turbo 15 RC 27

 Vivaspin® 20

5 to 20 mL samples The retentate volume is easily

Vivaspin® 20 centrifugal concentrators monitored using printed graduations
have been developed to offer and the modified dead-stop pocket
increased sample and process simplifies direct retentate retrieval.
flexibility and high performance.
In addition, unique accessories are
Featuring twin vertical membra nes for available for Vivaspin® 20, enabling
high filtration speeds, Vivaspin® 20 can pressurized, ultrafiltration and
achieve in excess of 100-fold constant volume diafiltration.
concentrations factors.

Technical Specifications
15

10
Concentrator capacity
5
Swing bucket rotor 20 mL
1
0.75
Fixed angle rotor 14 mL
116 0.5

0.2
With pressure head 15 mL

Dimensions

Length x diameter 116 x 30 mm

125 x 30 mm with pressure head

Active membrane area 6.0 cm2

Hold-up volume, membrane < 20 μL

30
Dead-stop volume 50 μL

Materials of construction

Body Polycarbonate (PC)

Filtrate vessel Polycarbonate (PC)

Concentrator cap Polypropylene (PP)

Pressure head Polyoxymethylene (POM) and

Aluminium (ALU)

Membrane Polyethersulfone (PES)

Equipment Required

Centrifuge

Rotor type Swing bucket Fixed angle (min. 25°)

Rotor cavity To fit 50 mL (30 mm) To fit 50 mL (30 mm)

conical bottom tubes conical bottom tubes

Maximum RCF 4,000 g 6,000 g

Maximum RCF, 3,000 g 6,000 g

≥100 kDa MWCO

Pressure

Pressure accessories VCA002, VCA005 and VCA200

Maximum pressure 5 bar (75 psi)

Maximum RCF, pressure-fuge* 3,000 g

Maximum RCF, pressure-fuge, 2,000 g

≥100 kDa MWCO*

*Swing bucket only

28 Protein Concentration Centrifugal Filtration Vivaspin® 20

Equipment Required (Continued)

Concentrate recovery

Pipette type Fixed or variable volume Fixed or variable volume

Recommended tip Thin gel loader type Thin gel loader type

Typical Performance Characteristics

Time to concentrate up to 30×

at 20°C and solute recovery

Mode Centrifuge Centrifuge Bench top Press-fuge

Rotor Swing bucket 25° Fixed angle Pressure Swing bucket

Centrifugal force | pressure 3,000 g 6,000 g 4 bar 3,000 g + 4 bar

Start volume 20 mL 14 mL 10 mL 10 mL

Min. Rec. Min. Rec. Min. Rec. Min. Rec.

Cytochrome c 0.25 mg/mL

(12.4 kDa)
3 kDa MWCO PES 110 97% 180 96% 60 96% – –

BSA 1.0 mg/mL (66 kDa)

5 kDa MWCO PES 23 99% 29 99% 50 98% 14 98%
10 kDa MWCO PES 16 98% 17 98% 32 97% 8 97%
30 kDa MWCO PES 13 98% 15 98% 32 97% 8 97%

IgG 0.25 mg/mL (160 kDa)

30 kDa MWCO PES 27 97% 20 95% 46 94% 13 97%
50 kDa MWCO PES 27 96% 22 95% 46 93% 13 96%
100 kDa MWCO PES 25 91% 20 90% 42 88% 12 94%

Latex beads 0.004% in DMEM

+10% FCS (55 nm)
300 kDa MWCO PES 20 99% 35 99% 10 99% – –

Latex beads 0.004% in DMEM

+10% FCS (240 nm)
1,000 kDa MWCO PES 4 99% 12 99% 4 99% – –

Yeast 1.0 mg/mL (S. Cerevisiae)

0.2 μm PES 15 95% 5 95% 20 95% 2 95%

Ordering Information

Vivaspin® 20 PES 12 pc 48 pc

3 kDa MWCO VS2091 VS2092

5 kDa MWCO VS2011 VS2012

10 kDa MWCO VS2001 VS2002

30 kDa MWCO VS2021 VS2022

50 kDa MWCO VS2031 VS2032

100 kDa MWCO VS2041 VS2042

300 kDa MWCO VS2051 VS2052

1,000 kDa MWCO VS2061 VS2062

0.2 μm VS2071 VS2072

Protein Concentration Centrifugal Filtration Vivaspin® 20 29

 Vivaspin® 100

20 to 98 mL samples Vivaspin® 100 units can also be used

Vivaspin® 100 bridges the gap for single or extremely sensitive
between centrifugal concentrators ­samples of up to 98 mL when
and crossflow cassettes. These devices pressurized and left on the bench, or
feature vertical membranes for high for temperature-­sensitive samples,
speed processing of even high particle placed into a refrigerator.
loaded samples. In addition, a unique Pressurization is made easy by use of
choice between centrifugal, pressure quick-release connectors and can be
or pressure-shake operating methods combined with orbital shaking for even
provides unrivaled process flexibility. faster sample concentration.

Fitting swing bucket rotors accepting In whichever mode Vivaspin® 100 is

250 mL bottles, Vivaspin® 100 offers used, the vertical membrane design
the highest sample capacity available inhibits membrane fouling while the
in a centrifugal device – up to an integrated dead-stop impedes
­astonishing 90 mL. concentration to dryness and loss of
sample.

Technical Specifications

Concentrator capacity
Swing bucket rotor 90 mL
Device fits standard 250 mL rotors With pressure head 98 mL
Dimensions
Length x diameter 123 x 62 mm
197 x 62 mm with pressure head
Active membrane area 23.5 cm2
Hold-up volume of membrane < 250 μL
Dead-stop volume 350 μL

Materials of construction
Body Polycarbonate (PC)
Filtrate vessel Polycarbonate (PC)
Concentrator cap Polypropylene (PP)
Pressure head Polyoxymethylene (POM) and Aluminium
(ALU)
Pressure head seal Thermoplastic Elastomer (TPE)
Membrane Polyethersulfone (PES)

Equipment Required
Centrifuge

Rotor type Swing bucket

Rotor cavity To fit 250 mL (60 mm) centrifuge bottles

(maximum cavity depth 105 mm)
Maximum RCF 2,000 g

Pressure

Pressure accessories VCA002, VCA800

Maximum pressure 5 bar (75 psi)

30 Protein Concentration Centrifugal Filtration Vivaspin® 100

---
Centrifuge
Process convenience
--
Pressure
Highest process
---
Pressure-shake
High process control

-
Low shear, no foaming control Ideal for single samples
Less visual control Use in fridge or cold Faster concentrations
room
Slower concentrations

Typical Performance Characteristics

Time to concentrate up to 30× at 20°C

90 mL start volume Swing bucket, Pressure, 4 bar (60 psi) Solute

2,000 g recovery
Static Orbital shake

BSA 1.0 mg/mL (66 kDa)

5 kDa MWCO PES 22 min 75 min 25 min 96%
10 kDa MWCO PES 16 min 60 min 20 min 96%
30 kDa MWCO PES 16 min 60 min 20 min 94%

IgG 0.25 mg/mL (160 kDa)

50 kDa MWCO PES 20 min 70 min 30 min 94%
100 kDa MWCO PES 20 min 85 min 30 min 90%

Latex beads 0.004% in DMEM

+ 10% FCS (55 nm)
300 kDa MWCO PES 35 min – 120 min 99%

Latex beads 0.004% in DMEM

+ 10% FCS (240 nm)
1,000 kDa MWCO* PES 4 min 5 min 4 min 99%

Ordering Information

Vivaspin® 100 PES with PP cap 2 pc 10 pc

5 kDa MWCO VC1011 VC1012

10 kDa MWCO VC1001 VC1002

30 kDaMWCO VC1021 VC1022

50 kDa MWCO VC1031 VC1032

100 kDa MWCO VC1041 VC1042

300 kDa MWCO VC1051 VC1052

1,000 kDa MWCO VC1061 VC1062

0.2 μm VC1071 VC1072

Protein Concentration Centrifugal Filtration Vivaspin® 100 31

 Vivaspin® Equipment and Accessories
Gas Pressure Ultrafiltration Constant Volume Diafiltration
When an appropriate centrifuge is In this procedure following
unavailable, or for single sample concentration, a diafiltration cup
processing, Vivaspin® 20 and 100 inserted into the Vivaspin® 20
centrifugal concentrators may be concentrator body is filled with buffer
pressurized with compressed gas for and centrifuged once to achieve 98%
bench-top concentration. salt removal. This compares to the
need for two centrifugation steps to
For even faster processing of samples achieve the same result with the re-fill
in Vivaspin® 20, gas pressure can be and re-spin approach for
combined with centrifugal force. This discontinuous diafiltration.
pressure-fugation method is
particularly suitable for difficult to filter The improved performance is due to
or viscous samples, such as serum, or the constant washing action of the
when using low process temperatures, exchange buffer from the diafiltration
which reduce filtration speed, and cup, as it replaces the original solvent
generally when minimum process time and salts when they pass through the
is essential. In a similar way, Vivaspin® ultrafiltration membrane.
100 may be pressurized and placed on
an orbital shaker for faster processing.

0.2

0.5
0.75
1

10

15
0.75

0.75
10
15
0.5
0.2

10

0.5
15

0.2
5
5
1

1
15

10

1
0.75
0.5

0.2

15

10

Spin or stand in rack

0.75
0.5
15
0.2

10

1
0.75
0.5

0.2

15 15

10 10

Pressurize
5 5

1 1
0.75 0.75
0.5 0.5

0.2 0.2

Using the Vivaspin® 20 pressure cap

32 Protein Concentration Pressure Filration Vivaspin® Equipment

Ordering Information

Vivaspin® Equipment and Accessories Pack Size Prod. No.

Air pressure controller (APC) fitted with pressure gauge, 1 VCA002
regulator, over-pressure safety valve and female
coupling. APCis supplied with extension line (4 mm
pneumatic tubing, 1 m) with male and female couplings,
and inlet tubing (6 mm pneumatic tubing, 1 m)
Charge valve for pressure head VCA200 1 VCA005
Female coupling 1 VCA010
Male coupling 1 VCA011
Replacement extension line 1 VCA012
(4 mm pneumatic tubing, 3 m)
Vivaspin® 20 pressure head 1 VCA200
Vivaspin 100 pressure head with seals
®
1 VCA800
Vivaspin 100 pressure head seals
®
10 VCA014
Diafiltration cups 12 VSA005

Protein Concentration Pressure Filtration Vivaspin® Equipment 33

 Vivaflow® 50

0.1 to 3 L

-
Unique performance

-
The novel Vivaflow® 50 system A single 50 cm2 module will typically
provides a standard of ease of use, reduce 500 mL to less than 15 mL in
performance, flexibility and economy under 50 minutes.

--
which is unrivalled by any laboratory Less than 10 mL minimum system
or pilot scale filtration system on the recirculation for highest

-
market. concentrations.
Less than 500 μL non
Unique features recoverablehold up volume.
Thin channel flip-flow path provides Near total recoveries achievable with

--
high turbulence and cross flow a single 10 mL rinse.
velocities for exceptional flux, even at
high concentrations. Each package of two cassettes

--
No need for pressure holders. contains all of the required tubing and
Crystal clear for simple control and fittings for plug-and-play operation
visibility of membrane status. with a standard peristaltic pump
Unique interlocking modules with accepting 6.4 mm OD (size 16) tubing.
series connectors for easy scale up.
Disposable | single use.
Single cassette

Technical Specifications

Dimensions

Overall L | W | H 25 | 107 | 84 mm

Channel W | H 15 mm | 0.3 mm

Active membrane area 50 cm2

Minimum recirculation volume < 10 mL

Multiple cassettes Hold-up volume, cassette 1.5 mL

Non recoverable hold-up < 0.5 mL

Operating conditions

Pump flow rate 200 – 400 mL/min

Maximum pressure 3 bar (45 psi)

Maximum temperature 60°C

Materials of construction

Main housing Polycarbonate (PC)

Flow channel Polymethylpentene (PMP)

Membrane Polyethersulfone (PES)

Regenerated Cellulose (RC)

Membrane support Polymethylpentene (PMP)

Seals and O rings Silicone (SIL)

Flow restrictor Polypropylene (PP)

Fittings Polyamide (PA)

Tubing Polyvinyl Chloride (PVC), medical grade

34 Protein Concentration Tangential Flow Filtration Vivaflow® 50

Typical Performance Characteristics

Time to concentrate up to 20x at 3 bar

inlet pressure, 20°C

Single device Three devices Solute recovery

250 mL 1L
Direct 10 mL rinse
start volume start volume

BSA 1.0 mg/mL (66 kDa)

5 kDa MWCO PES 34 min 49 min 96% > 99%
10 kDa MWCO PES 22 min 32 min 94% > 99%
30 kDa MWCO PES 22 min 32 min 92% 99%
50 kDa MWCO PES 20 min 29 min 92% 98%

IgG 1.0 mg/mL (160 kDa)

100 kDa MWCO PES 43 min 62 min 92% 98%
100 kDa MWCO RC 40 min 58 min 92% 98%

Yeast 1.0 mg/mL (S. Cerevisiae)

0.2 μm PES 33 min 47 min 92% 98%

Ordering Information

Vivaflow® 50 PES 2 cassettes

3 kDa MWCO VF05P9
5 kDa MWCO VF05P1
10 kDa MWCO VF05P0
30 kDa MWCO VF05P2
50 kDa MWCO VF05P3
100 kDa MWCO VF05P4
1,000 kDa MWCO VF05P6
0.2 μm VF05P7
Vivaflow 50 RC
®

100 kDa MWCO VF05C4

Protein Concentration Tangential Flow Filtration Vivaflow® 50 35

 Vivaflow® 50R

0.1 to 1 L samples Each cassette is supplied with all the

Concentrate 100 mL to under 20 mL tubing and a pressure indicator for
in just a few minutes or concentrate running the device with a laboratory
one liter 50 times in less than 60 pump and a size 16 pump head. For

--
minutes. Alternatively, speed up your speeding up concentration, two
process by using two Vivaflow® 50R cassettes can be run simultaneously.
cassettes in parallel and concentrate

--
1 liters in under 30 min. Fast and easy protein sample
concentration
Vivaflow® 50R is a plug-and-play Reusable
laboratory crossflow cassette for Concentrates volumes from

-
concentrating up to 1 L aqueous 0.1 L to 1 L
samples. The active membrane area Optimal for concentration of culture
per device is 50 cm2. supernatants and viruses
The most compact crossflow
cassette with a premium Hydrosart®
membrane

Vivaflow® 50R – Single cassette Technical Specifications

Dimensions

Overall L | W | H 24 | 100 | 100 mm

Channel W | H 7.5 | 0.4 mm

Active membrane area 50 cm2

Minimum recirculation volume 10 mL

Hold-up volume, cassette 1.7 mL

Non-recoverable hold-up < 0.5 mL

Operating conditions
Vivaflow 50R – Two cassettes
®
Pump flow rate 200 – 400 mL/min

Maximum pressure 4 bar (60 psi)

Maximum temperature 60°C

Materials of construction

Main housing Acrylic

Flow channel Acrylic

Membrane Hydrosart® (HY)

Membrane support Polyethylene (PE)

Seals and O-rings Silicone (SIL)

Pressure indicator Polypropylene (PP), SS spring

Flow restrictor Polypropylene (PP)

Fittings Polyamide (PA)

Tubing Polyvinyl Chloride (PVC), medical grade

36 Protein Concentration Tangential Flow Filtration Vivaflow® 50R

Visit us at Typical Performance Characteristics
www.sartorius.com/
Vivaflow50R Time to concentrate up to 20× at
for further information. 3.0 bar inlet | 2.5 bar outlet pressure, 20°C

-
Here you can find Start volume Average flux Recovery
instructions on how to 250 mL mL/min
Direct 25 mL rinse
use Vivaflow® 50R for:

-
Lysozyme 0.25 mg/mL (14.3 kDa)
Preparation of biological
5 kDa MWCO HY 70 3.4 96% 98%
nanoparticles and medical 10 kDa MWCO HY 23 10.3 94% 96%
nanocarriers
BSA 1.0 mg/mL (66 kDa)
Concentration and 10 kDa MWCO HY 24 9.9 98% >99%
purification of viruses 30 kDa MWCO HY 15 15.8 97% >99%

IgG 1.0 mg/mL (160 kDa)

100 kDa MWCO HY 46 5.2 97% >99%

Start volume 1 L (one Vivaflow 50R

®

at 3 bar), BSA 1.0 mg/mL

10 kDa MWCO HY 95 10.0 98% >99%

Start volume 1 L (two Vivaflow 50R

®

in parallel at 3 bar), BSA 1.0 mg/mL

10 kDa MWCO Hydrosart® 48 19.8 98% >99%

Ordering Information

Vivaflow® 50R HY 1 cassette

5 kDa MWCO VF05H1
10 kDa MWCO VF05H0
30 kDa MWCO VF05H2
100 kDa MWCO VF05H4

Protein Concentration Tangential Flow Filtration Vivaflow® 50R 37

 Vivaflow® 200

0.5 to 5 L Each cassette is supplied complete

Concentrate 250 mL to under 20 mL with all required tubing and a pressure
in just a few minutes or concentrate indicator. All you need is a peristaltic
one litre 50 times in less than 30 pump capable of handling 6.4 mm OD
minutes. Alternatively, use two (size 16) tubing. Should your pump
Vivaflow® 200 cassettes in parallel and head require larger tubing, link your
concentrate 5 litres in under 75 own tubing up to the standard
minutes. product, using the provided stepped
hose barb connector.
Near total sample recoveries can be
expected with most solutions. Two cassettes in parallel will
concentrate 5 litres in under 75
minutes.

Technical Specifications

Dimensions

Overall L | H | W 38 | 126 | 138 mm

Vivaflow® 200 set-up for
Channel W | H 10 mm | 0.4 mm
diafiltration
Active membrane area 200 cm2

Minimum recirculation volume < 20 mL

Hold up volume, cassette 5.3 mL

Non-recoverable hold-up < 2 mL

Operating conditions

Pump flow 200–400 mL/min

Maximum pressure 4 bar (60 psi)

Maximum temperature 60°C

Materials of construction

Main housing Acrylic

Flow channel Acrylic

Membrane Polyethersulfone (PES)

Hydrosart® (HY)

Membrane support Polypropylene

Seals and O rings Silicone (SIL)

Pressure indicator Polypropylene (PP), SS spring

Flow restrictor Polypropylene (PP)

Fittings Polyamide (PA)

Tubing Polyvinyl Chloride (PVC), medical grade

38 Protein Concentration Tangential Flow Filtration Vivaflow® 200

 Typical Performance Characteristics

Time to concentrate up to 20×

at 3 bar inlet pressure, 20˚C

1 litre Average flux Recovery

start volume mL/min
Direct 25 mL rinse

Lysozyme 0.25 mg/mL (14.3 kDa)

2 kDa MWCO HY 160 6 97% > 99%
3 kDa MWCO PES 180 5 97% > 99%
Operation – Single cassette
BSA 1.0 mg/mL (66 kDa)
5 kDa MWCO PES 29 33 98% > 99%
5 kDa MWCO HY 70 14 98% > 99%
10 kDa MWCO PES 23 41 96% > 99%
10 kDa MWCO HY 35 27 98% > 99%
30 kDa MWCO PES 25 38 96% 99%
30 kDa MWCO HY 20 48 96% > 99%
50 kDa MWCO PES 22 43 96% 98%

IgG 1.0 mg/mL

(160 kDa)
100 kDa MWCO PES 54 18 96% 99%

Yeast 1.0 mg/mL (S. Cerevisiae)

Operation – Two cassettes 0.2 μm PES 11 86 92% 98%

Dilute solute concentration, start volume

1 litre at 3 bar, 10 kDa MWCO PES
BSA 0.001 mg/mL 18 52 90% 98%
BSA 0.01 mg/mL 20 47 92% 98%
BSA 0.1 mg/mL 21 45 94% 99%

Start volume 5 litres (two VF200 in

parallel at 3 bar) 10 kDa MWCO PES
BSA 1.0 mg/mL (66 kDa) 67 70 97% > 99%

Ordering Information

Vivaflow® 200 PES 1 cassette

3 kDa MWCO VF20P9

5 kDa MWCO VF20P1

10 kDa MWCO VF20P0

30 kDa MWCO VF20P2

50 kDa MWCO VF20P3

100 kDa MWCO VF20P4

0.2 μm VF20P7

Vivaflow 200 HY
®

2 kDa MWCO VF20H9

5 kDa MWCO VF20H1

10 kDa MWCO VF20H0

30 kDa MWCO VF20H2

100 kDa MWCO VF20H4

Protein Concentration Tangential Flow Filtration Vivaflow® 200 39

 Vivaflow® Equipment

Pumps and Complete Systems transfer, in with precise flow control in

Masterflex® peristaltic pump drives and either direction. These pumps are also
heads perfectly complement Vivaflow® enabled for remote start | stop via a
crossflow cassettes, while also offering DB9 connector.
the flexibility, durability and
performance needed in multiple Loading tubing into the pump heads is
laboratory fluid transfer applications. fast and easy, thanks to the single-
With samples confined to single use thumb operated lever and automatic
tubing, there is no risk of cross- tubing retention. Each model is
contamination and no cleaning compatible with multiple tubing sizes,
required between each use. extending their suitability to further
applications, and the hard-wearing
Pump drives are offered for 230 or 115 V materials ensure long-term durability.
mains supplies. A variable speed,
reversible motor enables easy sample

Technical Specifications

Dimensions Masterflex® pump drives Easy-Load pump heads

Length x width x height 230 x 183 x 134 mm 64 x 105 x 105 mm

Weight 4.1 kg

Power output 37 W -

Speed | flow rate range 20 – 600 rpm 16 – 480 mL/min (size 16 tubing)
34 – 1,020 mL/min (size 15 tubing)

Materials of construction

Housing Polyarylamide (PARA) Polyarylamide (PARA)

Rotor - Stainless Steel (SS) and PTFE

Rollers - Stainless Steel (SS)

Ordering Information

Vivaflow® 50 System Pack Size Prod. No.

Pump drive (230 V), Easy-Load pump head (size 16), 500 mL 1 VFS502
diafiltration reservoir, cassette stand, spare tubing, T-connectors, series
interconnectors and pressure indicator

Pump drive (115 V), Easy-Load pump head (size 16), 500 mL diafiltration 1 VFS504
reservoir, cassette stand, spare tubing, T-connectors, series
interconnectors and pressure indicator

Vivaflow® 50R | 200 System Pack Size Prod. No.

Pump drive (230 V), Easy-Load pump head (size 16), 500 mL 1 VFS202
diafiltration reservoir, and spare tubing

Pump drive (115 V), Easy-Load pump head (size 16), 500 mL diafiltration 1 VFS204
reservoir, and spare tubing

Pump Drives and Heads Pack Size Prod. No.

Masterflex® Economy Drive variable speed peristaltic pump (230 V) 1 VFP001

Masterflex Economy Drive variable speed peristaltic pump (115 V)

®
1 VFP002

Masterflex Easy-Load pump head (Size 16)

®
1 VFA012

Masterflex Easy-Load pump head (Size 15)

®
1 VFA013

40 Protein Concentration Tangential Flow Filtration Vivaflow® Equipment

Vivaflow® Accessories

Scaling Up first concentrated to the desired

The maximum throughput or speed of volume, then a length of tubing placed
filtration with Vivaflow® cassettes can into a separate vessel containing the
be easily increased, thanks to the exchange buffer is connected to the
modular design. Up to 6x Vivaflow® 50 reservoir. Airtight sealing in the lid
cassettes can be operated in series and enables constant volume diafiltration.
parallel, while two Vivaflow® 50R or 200 As the original buffer and salts
cassettes may be run in parallel. permeate the ultrafiltration membrane,
they are replaced by an equal volume
Accessories and Replacement Parts of exchange buffer, thereby avoiding
Optional accessories such as the the large buffer volumes and possibility
diafiltration reservoir, make of sample dilution, which can be
concentration and diafiltration common to alternative methods.
exceptionally convenient. A sample is

Technical Specifications

Materials of construction

Cassette stand Aluminium (ALU)

Tubing Polyvinyl Chloride (PVC), medical grade

Luer fittings Polyamide (PA)

Flow restrictors Polypropylene (PP)

Pressure indicator Polypropylene (PP), Stainless Steel (SS), Polyoxymethylene

(POM), Silicone (SIL) and Polyamide (PA)

Diafiltration reservoir Polycarbonate (PC), Polyoxymethylene (POM), Silicone (SIL),

Polyvinyl Chloride (PVC) and Polyamide (PA)

Ordering Information

Vivaflow® accessories for operating multiple cassettes Pack Size Prod. No.

T-connector (for 2-6x Vivaflow 50 | 2x Vivaflow 50R)

® ®
2 VFA030

Series interconnector (for 2-6x Vivaflow 50)

®
6 VFA031

Y-connector (size 15 to 2x size 16, for 2x Vivaflow 200)

®
1 VFA005

Vivaflow accessories and replacement parts

®

Vivaflow® 50 cassette stand 1 VFA016

500 mL diafiltration reservoir 1 VFA006

Pump tubing, 3 m, with luer fittings (size 16) 1 VFA004

Pump tubing, 3 m, with luer fittings (size 15) 1 VFA003

Female Luer lock to hose barb fittings (size 16) 10 VFA032

Female Luer lock to hose barb fittings (size 15) 10 VFA036

Flow restrictors, 0.6 mm 6 VFA035

Flow restrictors, 0.4, 0.6 and 0.8 mm (2 of each) 6 VFA009

Pressure indicator (1-3 bar) 1 VFA020

Vivaflow® tubing set (2x 1 m feed tubing with Luer fitting, 2x 0.5 m 1 VFA034
retentate tubing with flow restrictor, 1x series interconnector)

Protein Concentration Tangential Flow Filtration Vivaflow® Accessories 41

 Ultrafiltration Membrane Discs

Polyethersulfone (PES) membrane support that could trap or

This is a general purpose membrane bind passing microsolutes, these
that provides excellent performance membranes are preferred for sample
with most solutions when high cleaning, ­protein removal, and when
­recoveries in the retentate are of high recoveries from the filtrate are of
­primary importance. Polyethersulfone primary ­importance.
membranes exhibit no hydrophobic
or hydrophillic interactions and are Hydrosart® (HY)
usually preferred for their low fouling These membranes are also highly
characteristics, exceptional flux and hydrophillic and are often preferred
broad pH compatibility. for their higher protein recovery
when processing very dilute solutions.
Cellulose Triacetate (CTA) Resistance to autoclaving, ease of
High hydrophilicity and very low non- cleaning and extended chemical
specific binding characterize this compatibility also characterize this
membrane. Cast without any ­membrane material.

Technical Specifications and Typical Performance

Polyethersulfone, Type Cellulose Triacetate, Hydrosart®, Type 144

146 Type 145

Thickness 120 μm 120 μm 180 μm

pH range 1 – 14 4–8 1 – 13

Flux with water

10 kDa MWCO 0.2 mL/min/cm2 0.11 mL/min/cm2 0.08 mL/min/cm2

Protein retention

Cytochrome c 95% 90% 99%

Ordering Information

PES Membrane Discs, Type 146 Diameter 10 pc

1 kDa MWCO 47 mm 14609--47------D

63 mm 14609--63------D

76 mm 14609--76------D

5 kDa MWCO 25 mm 14629--25------D

47 mm 14629--47------D

63 mm 14629--63------D

76 mm 14629--76------D

10 kDa MWCO 25 mm 14639--25------D

43 mm 14639--43------D

47 mm 14639--47------D

63 mm 14639--63------D

76 mm 14639--76------D

150 mm 14639-150------D

42 Protein Concentration Ultrafiltration Membrane Discs

PES Membrane Discs, Type 146 (con't) Diameter 10 pc

30 kDa MWCO 25 mm 14659--25------D

47 mm 14659--47------D

63 mm 14659--63------D

76 m 14659--76------D

50 kDa MWCO 25 mm 14650--25------D

47 mm 14650--47------D

76 mm 14650--76------D

100 kDa MWCO 47 mm 14668--47------D

63 mm 14668--63------D

300 kDa MWCO 25 mm 14679--25------D

47 mm 14679--47------D

76 mm 14679--76------D

CTA Membrane Discs, Type 145

5 kDa MWCO 25 mm 14529--25------D

47 mm 14529--47------D

10 kDa MWCO 25 mm 14539--25------D

47 mm 14539--47------D

50 mm 14539--50------D

20 kDa MWCO 25 mm 14549--25------D

43 mm 14549--43------D

47 mm 14549--47------D

63 mm 14549--63------D

HY Membrane Discs, Type 144

2 kDa MWCO 47 mm 14419--47------D

63 mm 14419--63------D

5 kDa MWCO 25 mm 14429--25------D

44 mm 14429--44------D

47 mm 14429--47------D

63 mm 14429--63------D

76 mm 14429--76------D

10 kDa MWCO 25 mm 14439--25------D

47 mm 14439--47------D

63 mm 14439--63------D

76 mm 14439--76------D

30 kDa MWCO 25 mm 14459--25------D

47 mm 14459--47------D

63 mm 14459--63------D

76 mm 14459--76------D

100 kDa MWCO 47 mm 14468--47------D

Protein Concentration Ultrafiltration Membrane Discs 43

44 Chapter Site Title
DNA Concentration
Table of Contents

Vivacon® 500 46

Vivacon® 2 49

45
 Vivacon® 500

100 to 500 μL samples Vivacon® 500 PCR Grade

Vivacon® 500 centrifugal When using DNA amplification
concentrators offer the optimal technologies, any traces of DNA
solution for DNA and protein originating from the equipment have
concentration and buffer exchange to be eliminated.
applications. For optimal performance
with very dilute samples, e.g. forensic Vivacon® 500 PCR Grade units are
samples, Vivacon® 500 is equipped treated with ethylene oxide (ETO)
with the patented regenerated in a validated process in order to
cellulose membrane, Hydrosart®. deactivate all traces of DNA that
might interfere with subsequent
High recoveries and excellent amplification procedures.
reproducibilities are paired with
convenience offered by molecular Ref.: K. Shaw et al., Int. J. Legal Med.
weight cut-off printed on individual (2008) 122: 29–33
devices.

The possibility of a reverse-spin after

sampleprocessing assures complete
con­centrate recovery which is
especially important when working
with low sample concentrations.

Feature Benefit

Reverse-spin enabled Complete and highly reproducible

sample recovery

Low binding material High recoveries of low sample concentrations

Reverse spin
Technical Specifications

Concentrator capacity

Fixed angle rotor 0.5 mL

Dimensions

Length x diameter 45 x 12.4 mm

47.5 x 12.4 mm reverse spin

Active membrane area 0.32 cm2

Hold-up volume, of membrane and support < 5 μL

Dead-stop volume (40° rotor) 5 μL

Materials of construction

Body Polycarbonate (PC)

Filtrate vessel Polypropylene (PP)

Membrane Hydrosart® (HY)

Cellulose Acetate (CA)

46 DNA Concentration Vivacon® 500

Conversion Table for MWCO to Nucleotide Cut-Off

Membrane Single-Stranded Double-Stranded

Cut-Off (SSCO) Cut-Off (DSCO)

2 kDa HY > 10 bases > 10 bp

10 kDa HY > 90 bases > 30 bp

30 kDa HY > 275 bases > 50 bp

50 kDa HY > 475 bases > 300 bp

100 kDa HY > 900 bases > 600 bp

125 kDa CA > 1,000 bases > 650 bp

Typical Performance Characteristics for DNA

Start volume 0.5 mL, sample concentration 50 ng/mL

MWCO Nucleic Acid Time to Concentrate up Concentrate RCF

Length to 30× at 20°C Recovery

2 kDa 10 bp 60 min 93% 7,500 g

10 kDa 30 bp 25 min 94% 7,500 g

30 kDa 50 bp 18 min 88% 5,000 g

50 kDa 300 bp 18 min 91% 5,000 g

100 kDa 600 bp 10 min 87% 3,000 g

125 kDa 650 bp 12 min 85% 2,000 g

125 kDa 900 bp 9 min 94% 3,000 g

Typical Performance Characteristics for Proteins

Start volume 0.5 mL, sample and concentration of proteins as specified in table

MWCO Test Molecule Time to Concentrate up Concentrate RCF

to 30× at 20°C Recovery

2 kDa 0.25 mg/mL 30 min 95% 14,000 g

cytochrome c

10 kDa 0.25 mg/mL 15 min 92% 14,000 g

cytochrome c

30 kDa 1.0 mg/mL BSA 10 min 95% 14,000 g

50 kDa 1.0 mg/mL BSA 10 min 92% 14,000 g

100 kDa 1.0 mg/mL 11 min 90% 8,000 g

bovine IgG

125 kDa 1.0 mg/mL 10 min 81% 8,000 g

bovine IgG

DNA Concentration Vivacon® 500 47

 Ordering Information

Vivacon® 500 25 pc 100 pc 500 pc

2 kDa MWCO VN01H91 VN01H92 -

10 kDa MWCO VN01H01 VN01H02 -

30 kDa MWCO VN01H21 VN01H22 VN01H23

50 kDa MWCO VN01H31 VN01H32 VN01H33

100 kDa MWCO VN01H41 VN01H42 VN01H43

125 kDa MWCO VN01H81 VN01H82 VN01H83

Vivacon® 500 PCR

Grade

30 kDa MWCO - VN01H22ETO VN01H23ETO

100 kDa MWCO - VN01H42ETO -

125 kDa MWCO - VN01H82ETO VN01H83ETO

Vivacon® 500 Accessories

Additional collection tubes, 100 pc VNCT01

48 DNA Concentration Vivacon® 500

 Vivacon® 2

0.4 – 2 mL samples The possibility of a re-spin after sample

Vivacon® 2 centrifugal concentrators processing assures complete con­
offer the optimal solution for DNA centrate recovery which is especially
and protein concentration and buffer important when working with low
exchange applications. For optimal sample concentrations.
performance with very dilute samples,
e.g. forensic samples, Vivacon® 2 is Vivacon® 2 PCR Grade
equipped with the patented regener- Vivacon® 2 PCR Grade units are
ated cellulose membrane Hydrosart®. treated with ethylene oxide (ETO)
in a validated process in order to
High recoveries and excellent repro- deactivate all traces of DNA that
ducibilities are paired with conve- might interfere with subsequent
2.0
mL
nience offered by volume graduation amplification procedures.
and molecular weight cut-off printed
1.5

1.0

on individual devices.

Feature Benefit

Re-spin possibility Complete and highly reproducible

sample recovery

Low binding material High recoveries of low sample concentration

1.0
Easy to remove re-spin cap Convenient sample handling

Graduation printed on Optimal process control

1.5

mL
2.0

Technical Specifications

Concentrator capacity

Fixed angle rotor 2 mL

Reverse spin
Dimensions

Length x diameter 125 x 16 mm

115 x 16 mm reverse spin

Active membrane area 0.95 cm2

Hold-up volume, membrane and support 10 μL

Dead-stop volume (25° rotor) 55 μL

Materials of construction

Body Polycarbonate (PC)

Filtrate vessel Polypropylene (PP)

Reverse-spin recovery vial Polypropylene (PP)

Recovery vial cap Polypropylene (PP)

Membrane Hydrosart® (HY)

Cellulose Acetate (CA)

DNA Concentration Vivacon® 2 49

 Conversion Table for MWCO to Nucleotide Cut-Off

Membrane Single-Stranded Double-Stranded

Cut-Off (SSCO) Cut-Off (DSCO)

2 kDa HY > 10 bases > 10 bp

10 kDa HY > 90 bases > 30 bp

30 kDa HY > 275 bases > 50 bp

50 kDa HY > 475 bases > 300 bp

100 kDa HY > 900 bases > 600 bp

125 kDa CA > 1,000 bases > 650 bp

Typical Performance Characteristics for DNA

Volume 2 mL, sample concentration 50 ng/mL, start volume: 2 mL

MWCO Nucleic Acid Time to Concentrate up Concentrate RCF

Length to 30× at 20°C Recovery

2 kDa 10 bp 120 min 92% 7,500 g

10 kDa 30 bp 60 min 94% 5,000 g

30 kDa 50 bp 60 min 95% 2,500 g

50 kDa 300 bp 45 min 96% 2,500 g

100 kDa 600 bp 30 min 93% 2,500 g

125 kDa 650 bp 30 min 88% 2,500 g

125 kDa 900 bp 30 min 89% 2,500 g

Typical Performance Characteristics for Proteins

Start volume 2 mL, sample and concentration of proteins as specified in table

MWCO Test Molecule Time to Concentrate up Concentrate RCF

to 30× at 20°C Recovery

2 kDa 0.25 mg/mL 120 min 95% 7,500 g

cytochrome c

10 kDa 0.25 mg/mL 90 min 96% 5,000 g

cytochrome c

30 kDa 1.0 mg/mL BSA 40 min 96% 5,000 g

50 kDa 1.0 mg/mL BSA 30 min 94% 5,000 g

100 kDa 1.0 mg/mL 30 min 92% 5,000 g

bovine IgG

125 kDa 1.0 mg/mL 27 min 81% 5,000 g

bovine IgG

50 DNA Concentration Vivacon® 2

Ordering Information

Vivacon® 2 25 pc 100 pc 500 pc

2 kDa MWCO VN02H91 VN02H92 -

10 kDa MWCO VN02H01 VN02H02 -

30 kDa MWCO VN02H21 VN02H22 VN02H23

50 kDa MWCO VN02H31 VN02H32 -

100 kDa MWCO VN02H41 VN02H42 VN02H43

125 kDa MWCO VN02H81 VN02H82 VN02H83

Vivacon® 2 PCR Grade

30 kDa MWCO - VN02H22ETO -

50 kDa MWCO - VN02H32ETO -

100 kDa MWCO - VN02H42ETO VN02H43ETO

125 kDa MWCO - - VN02H83ETO

DNA Concentration Vivacon® 2 51

52 Chapter Site Title
Protein Purification
Table of Contents

Vivaclear Centrifugal Filters 54

Vivapure® Ion Exchange Purification Products 56

53
 Vivaclear Centrifugal Filters

Vivaclear centrifugal filters are dispos-

-
Applications

---
able microfiltration devices for the fast Clarification of samples before
and reliable clarification or filtration of loading onto Vivapure® protein
biological samples in the range 100 t­ o purification spin columns
500 μL. They can be used in fixed Removal of particles and participates
angle rotors accepting 2.2 mL centri- Filtration of plasma and serum

--
fuge tubes. Removal of cells or cell debris

Product Features

--
High-flux polyethersulfone
membrane
0.8 μm pore size
Low hold-up volume (< 5 μL)
Fast and reproducible performance

54 Protein Purification Vivaclear Centrifugal Filters

Technical Specifications

Filter capacity

Swing bucket rotor do not use

Fixed angle rotor 500 μL

Dimensions

Length x diameter 43 x 11 mm

Active membrane area 0.34 cm2

Hold-up volume, membrane and support < 5 μL

Materials of construction

Body Polypropylene (PP)

Filtrate tube Polypropylene (PP)

Membrane Polyethersulfone (PES)

Equipment Required

Centrifuge

Rotor type Fixed angle (40-45°)

Rotor cavity To fit 2.2 mL (11 mm) conical bottom tubes

Maximum RCF 2,000 g

Filtrate recovery

Pipette type Fixed or variable volume

Recommended tip Standard type

Ordering Information

Vivaclear Mini 100 pc

0.8 μm PES VK01P042

Protein Purification Vivaclear Centrifugal Filters 55

 Vivapure® Ion Exchange Purification Products

Fast and easy-to-use spin columns The microporous structure of these

Vivapure® Ion Exchange (IEX) devices membrane adsorbers has a pore size
incorporate Sartobind® membrane > 3 μm, which is orders of magnitude
­adsorber technology as the larger than conventional
chromatography matrix. The ready-to- chromatography resins. This allows
use spin column format makes protein molecules to be transported to the
­purification as easy as filtration. With ligands immobilized on the membrane
no risk of running dry, Vivapure® adsorber by convective flow,
replaces time-consuming and overcoming the diffusion limitations of
SEM comparing chromatography expensive r­ esin-based chromatography resins, and leading to
beads (right) with the Sartobind® chromatography in many protein very high flow rates. The large pore
membrane adsorber, which fea-
purification workflows. sizes also prevent gel filtration effects
tures 50x larger pore sizes.
and minimize non-specific binding.
The rapid bind-wash-elute protocol is
especially ideal in screening With Vivapure®, there is no need for
applications, where multiple samples column packing, saving time and
or purification conditions can be ­ensuring reproducibility. Furthermore,
conveniently processed in parallel. Sartobind® membrane adsorber
­technology is available in process scale
formats, making Vivapure® an
indispensible tool for process
development prior to purification
scale-up.

Spin Spin

Original Protein is bound Wash Elute Pure

sample to membrane protein

Fast and easy protein purification with Vivapure® spin columns

56 Protein Purification Vivapure® Ion Exchange Protein Purification Products

 Membrane Availability

Functional groups Ion exchanger type

Sulphonic acid (S) Strong acidic cation exchanger: R-CH₂-SO₃-Na+

Quaternary ammonium (Q) Strong basic anion exchanger: R-CH₂-N+-(CH₃)₃Cl-

Diethylamine (D) Weak basic anion exchanger: R-CH₂-NH+-(CH₂H₅)₂

Typical Performance Characteristics

Vivapure® Mini H
Vivapure® spin Protein binding Max. volume, Max. volume,
columns capacity* swing bucket fixed angle

Vivapure® Mini H 4 mg – 0.4 mL

Vivapure Maxi H
®
60 – 80 mg 19 mL 10.5 mL

---
Typical Applications

--
Fractionation of protein mixtures prior to 1D or 2D-PAGE
Scouting purification conditions for new protein targets
Removal of endotoxins from monoclonal antibodies

---
Vivapure® Maxi H Preparation of heme moiety from heme containing protein prior to functional
analysis
General protein purification and polishing

-
Detergent removal from protein solutions
Purification of antibodies from serum, ascites or cell culture supernatant
Intermediate sample purification prior to further HPLC | FPLC
Purification of membrane-bound proteins

Detailed application notes are available on our website: www.sartorius.com

Vivapure® Mini H Ordering Information

Vivapure® Mini Spin Centrifuge Prod. No.

Columns Tubes

Vivapure® D Mini H 24 48 VS-IX01DH24

Vivapure Q Mini H
®
24 48 VS-IX01QH24

Vivapure S Mini H
®
24 48 VS-IX01SH24

Vivapure Maxi
®

Vivapure® D Maxi H 8 16 VS-IX20DH08

Vivapure Q Maxi H
®
8 16 VS-IX20QH08
Vivapure® Maxi H
Vivapure S Maxi H
®
8 16 VS-IX20SH08

* Binding capacities established using

1 ­mg/mL BSA in 25 mM Tris-HCl (pH 8) for
Vivapure® Q and D devices, or 1 mg/mL
­cytochrome c in 25 mM sodium acetate
­buffer (pH 5.5) for Vivapure® S devices.
­Actual capacities depend on the target
­molecule and selected buffer conditions.

Protein Purification Vivapure® Ion Exchange Protein Purification Products 57

58 Chapter Site Title
Virus Purification and
Concentration
Table of Contents

Vivapure® Virus Purification and Concentration Kits 60

Adenovirus Purification 61

Vivapure® Adenopack 20 62

Vivapure® Adenopack 100 63

Vivapure® Adenopack 500 65

Lentivirus Purification 66

Vivapure® Lentiselect 40 67

Vivapure® Lentiselect 500 68

Vivapure® Lentiselect 1000 69

59
 Vivapure® Virus Purification and Concentration Kits

Recombinant virus vectors are the Rapid Virus Purification by

preferred method for a wide range of Membrane Chromatography
gene delivery applications. Especially The Sartobind® ion exchange
adenovirus type 5 and VSV-G membrane adsorber technology used
pseudotyped lentivirus are two in Adenopack and Lentiselect is
frequently utilized viral vectors for in unique in its capability to efficiently
vitro and in vivo applications. and ­rapidly capture and recover large
virus particles. When compared to
Recombinant Adenovirus Vectors chromatography media, membrane
Recombinant adenovirus vectors are adsorbers provide large 3000 nm
versatile tools in research and pores allowing unrestricted access
therapeutic applications for gene and recovery of virus from the charged
transfer and protein expression in cell adsorber surface. Convective flow
lines that have low transfection through the syringe filter devices
efficiency with liposomes. provides high-speed separations not
possible with traditional
After entering cells, the virus remains chromatography, cesium chloride
epichromosomal (i.e. does not density gradients and
integrate into the host chromosome, ultracentrifugation methods.
leaving the host genome unaffected).
The delivery of RNAi into cells is Our membrane adsorbers with porous
becoming a major application for matrices, high capacities, low differ­
adenovirus vectors. ential pressures, high flow rates and
low unspecific adsorption show an
Lentivirus Vectors excellent performance in small scale
Lentivirus vectors are frequently used virus purification. Additionally, they
in gene transfer studies, due to their are also scalable and confirm to
ability of gene transfer and integration cGMP facilities to large volume, high
into dividing and non-dividing cells. performance separation, reducing
The pseudotyped envelope with the processing time by a factor of 10
vesicular stomatitis virus envelope G in the final process.
(VSV-G) protein broadens their target
cell range. Lentiviral vectors have been
shown to deliver genes into cell types
(e.g. neurons, lymphocytes and
macrophages) which other retrovirus
vectors could not be used for. The
lentivirus vector is increasingly used
to integrate siRNA efficiently in a wide
variety of cell lines and primary cells,
both in vitro and in vivo.

60 Virus Purification and Concentration Vivapure® Virus Purification and Concentration Kits
 Adenovirus Purification

--
Adenopack 20 | 100 | 500 Adenopack Advantages
The Adenopack adenovirus purifi­
cation and concentration kits offer Fast and Easy Virus Purification
researchers who need to recover up to Purification completed in 2 hours
3 × 1013 purified recombinant adeno­ Convenient, over 10 × faster

-
virus particles for invitro transfection a alternative to CsCl density gradient
fast, safe and easy to use solution. The
kits include all reagents and devices Quantitative Yields
necessary for clarification, purification In contrast to CsCl density gradient,
and concentration of adenovirus the complete cell culture is used for
type 5 from HEK293 cell cultures in virus purification and not only the

-
only two hours. These straight forward viral pellet
kits replace time-consuming and
labor-intensive 48 hour CsCl density Flexible Product Range
gradients. Applicable from initial construct
screening to large scale virus

-
Adenopack kits are offered as production
Adenopack 20, Adenopack 100
and Adenopack 500, for the Complete Kit
purification and concentration of Including filtration devices,
adenovirus type 5 from 20 mL to Adenopack units for virus purifica-

-
500 mL cell culture, leading to tion, Vivaspin® and all buffers
1 × 1011- 3 × 1013 purified viral particles.
For each sample volume, the most Low Endotoxin Levels
convenient handling method is High cell viability and infection
offered for ultimate convenience. rates due to endotoxin levels of
< 0.025 EU/mL
To this end, preparations using
Adenopack 20 are pursued in
spin column format in a centrifuge,
Adenopack 100 is a manually
operated kit in syringe filter format*,
and Adenopack 500 is a pump
driven kit.

Purification of GFP-Ad5 Constructs

Purification method Process time Eluate Recovery*** Viral Particles

Adenopack 20 | 20 mL culture 1 hour 1 mL 65 – 70% 1 × 1011-12

Adenopack 100 | 60 mL culture 1 – 2 hours 1 mL 65% 1 – 3 × 1012

Adenopack 100 | 200 mL culture 2 hours 1 mL 80% 1 × 1013

Adenopack 500 | 500 mL culture 2 hours 1 mL 80% 1 – 3 × 1013

500 mL CsCl density gradient 24 – 48 hours 1 – 2 mL** 60 – 70% 1 × 1011-12

* Vivapure® Adenopack 100 can optionally

be operated with a laboratory pump and an
infusion pump, for which protocols are pro-
vided on our web page www.sartorius.com.
Additionally, the tubes and adaptors needed
for these operation modes can be ordered.
** after dialysis
*** before buffer exchange

Virus Purification and Concentration Adenovirus Purification with Vivapure® Adenopack Kits 61
 Vivapure® Adenopack 20
The optimal kit for construct screening

Vivapure® Adenopack 20 is the in the CsCl density gradient method –

­downscale kit in the Adenopack for the first time adenovirus type 5 can
series, purifying up to 1 × 1012 adeno­ efficiently be purified from less than
virus type 5 particles from 20 mL cell 100 mL cell culture volume!
culture. Especially when testing new
constructs, parallel and fast Typical Performance
purifications of different adenoviruses For a normal yielding vector, 1 × 15 cm
are essential. This kit allows the rapid, culture plate purified using this
simple and affordable spin column method yields up to 1 × 1012 viral
based purification of 6 different particles.
samples in parallel and bridges a gap

Ordering Information and Kit Contents

Vivapure® Adenopack 20 VS-AVPQ020

Vivapure Adenopack 20 RT*
®
VS-AVPQ022
Adenopack Maxi spin columns 6
Vivaclear Maxi 0.45 μm PES 6
Empty 50 mL tubes 6
Loading Buffer (10×) 25 mL
Washing Buffer (10×) 30 mL
Elution Buffer 20 mL
Benzonase (12.5 U/μL)
®
120 μL
Vivaspin 20, 100 kDa MWCO
®
6
Instructions 1 each for Kit and Vivaspin®

Technical Data

Kit specifications
Sample size 20 mL of cell culture
Number of purifications 6 × 20 mL
Virus particles (VP) per mL Typically up to 1 × 1011 – 1012
VP | IU 50–100
Processing time Typically 1 hour
Endotoxin level < 0.025 EU/mL

spin spin spin

* A  denopack 20 RT does not include

Benzonase®
** Benzonase® Nuclease is manufactured by
Merck KGaA, Darmstadt, Germany and
is covered by US Patent 5,173,418 and EP
Patent 0,229,866. Nycomed Pharma A/S 1. Treat with Benzonase®** 5. Bind virus and wash 6. Elute purified virus Final
(Denmark) claims worldwide patent rights 2. Filter with Vivaclear Maxi away contaminants concentration|
to Benzonase® Nuclease, which are licensed 3. Add 10× Loading Buffer buffer exchange
exclusively to Merck KGaA, Darmstadt, to sample
Germany. Benzonase® is a registered trade- 4. Equilibrate Adenopack Maxi spin columns
mark of Merck KGaA, Darmstadt, Germany.

62 Virus Purification and Concentration Vivapure® Adenopack 20

 Vivapure® Adenopack 100
Fast purification of up to 1 × 1013 viral particles

Vivapure® Adenopack 100 is optimally However, for even more convenience,

suited for adenovirus purification from protocols are provided for optionally
up to 200 mL cell culture for in vitro running the virus purification with a
transfection. This flexible kit contains peristaltic pump or with an infusion
two Adenopack 100 units, which pump, in additional to detailed
can be either used in tandem for the instructions for a manual operation
purification of up to 200 mL cell supplied with the kit. The accessories
culture for recovering 1 × 1013 viral needed for the operation with a pump
particles or individually for purifying are supplied as individual products.
1–3 × 1012 viral particles from up to
60 mL cell culture. The purification Typical Performance
is pursued manually with a syringe For a normal yielding vector, 10 × 15 cm
optimally attached to a retort stand. culture plate purified using this
method yields up to 1 × 1013 viral
particles.

Ordering Information and Kit Contents

Vivapure® Adenopack 100 VS-AVPQ101

Vivapure Adenopack 100 RT*

®
VS-AVPQ102

Adenopack 100 units 2

Minisart NML Plus

®
4

20 mL syringe 4

Tubing set and one way valve 2

10 mL syringe (elution) 2

Loading Buffer (10×) 1 × 25 mL

Washing Buffer 1 × 120 mL

Elution Buffer 1 × 20 mL

Benzonase 12.5 U/μL

®
200 μL

Vivaspin 20 concentrator
®
4

Instructions 1 each for Kit and Vivaspin®

Adenopack 100 Accessories

Pump tubing set for Vivapure® Adenopack 100 VS-AVPA001

* Adenopack 100 RT does not include

Benzonase®

Virus Purification and Concentration Vivapure® Adenopack 100 63

 Technical Data

Kit specifications
Sample size 20–200 mL of cell culture
Number of purifications 2 × 20–60 mL
1 × 200 mL
Virus particles (VP) per mL Typically up to 1 × 1013
VP | IU 20–50
Processing time Typically 2 hours
Endotoxin level < 0.025 EU/mL

1. Add Benzonase® 2. Load cleared 3. Wash 4. Elute Concentrate

and clarify sample sample

64 Virus Purification and Concentration Vivapure® Adenopack 100

 Vivapure® Adenopack 500
Pump driven kit for larger volumes

Vivapure® Adenopack 500 is the to use product replaces lengthy and

direct upscale kit to the Adenopack inefficient cesium chloride density
100, for adenovirus purification. In gradient methods.
only 2 hours up to 3 × 1013 adenovirus
particles are purified and concentrated Typical Performance
from 500 mL cell culture. This For a normal yielding vector, 25 × 15 cm
completely ready-to-use kit is culture plate purified using this
conveniently operated by a laboratory method yields up to 3 × 1013 viral
pump, offering optimal flow control particles.
and minimal hands-on time. This easy

Ordering Information and Kit Contents

Vivapure® Adenopack 500 VS-AVPQ501

Vivapure Adenopack 500 RT*
®
VS-AVPQ502
Adenopack 500 unit 1
Sartopore 2 150
®
1
Tubing set and one way valve 2
10 mL syringe 1
Loading Buffer (10×) 60 mL
Washing Buffer (10×) 30 mL
Elution Buffer 20 mL
Benzonase® 12.5 U/μL 500 μL
Vivaspin 20 concentrator
®
2
Instructions 1 each for Kit and Vivaspin®

Technical Data

Kit specifications
Sample size 500 mL of cell culture
Number of purifications 1 × 500 mL
Virus particles (VP) per mL Typically up to 3 × 1013
VP | IU 20–50
Processing time Typically 2 hours
Endotoxin level < 0.025 EU/mL

1. Add Benzonase® and 2. Load sample and wash 3. Elute 4. Concentrate

* Adenopack 500 RT does not include clarify sample unspecifically bound proteins
Benzonase®

Virus Purification and Concentration Vivapure® Adenopack 500 65

 Lentivirus Purification

-
Lentiselect 40 | 500 | 1000 Lentiselect Advantages
The Lentiselect lentivirus purification
and concentration kits offer Fast and Easy Virus Purification

-
researchers who need to recover up to Purification completed in under one
5 × 109 infective lentivirus particles per to six hours, depending on sample
mL for invitro transfection or animal volume

-
studies a fast and easy to use solution. Kit as easy to use as filtration

These straight forward kits replace No Need for Expensive Instruments

time-consuming ultracentrifugation Lentivirus purification with
protocols, which typically take ­Lentiselect is independent of

-
approximately one day for large sample equipment such as ultracentrifuges
volumes, thus reducing the purification
time to only a few hours. High Virus Purity
Achieve pure virus due to a
Lentiselect kits are offered as chromatography purification for your
Lentiselect 40, Lentiselect 500 and experiments instead of a crude and
Lentiselect 1000 for the purification variable cell culture supernatant
and concentration of VSV-G pellet

-
pseudotyped lentivirus from 40 mL to 1
L cell culture, leading to 8 × 108 – 1 × 1010 Optimal for Multiple Virus Construct
­purified infective particles. For each Screening
sample volume, the most convenient With Lentiselect 40, four purification
handling method is offered. To this runs can be conducted in parallel

-
end, 40 mL sample volumes are with one kit
processed manually with Lentiselect
40, while Lentiselect 500 and 1000 Complete Kits
are pump driven kits. Including Lentiselect units for
virus purification, Vivaspins for
concentration | buffer exchange and

-
all buffers and syrings necessary

Low Endotoxin Levels

High cell viability and infection
rates due to endotoxin levels of
< 0.025 EU/mL

Purification of VSV-G Pseudotyped Lentivirus Constructs

Purification method Process Eluate Viral Recovery Infective Viral

time Particles/mL Particles

Lentiselect 40   45 min 200 μL* 4 × 109 50% 8 × 108

40 mL sample

Lentiselect 500 3 hours 1 mL* 3 × 109 35% 2 – 5 × 109

500 mL sample

Lentiselect 1000   6 hours 2 mL* 5 × 109 35% 1 × 1010

1 L sample

Ultracentrifugation   10 – 11 hours 500 μL 6 × 109 25% 3 × 109

500 mL sample

* After desaltin | buffer exchange

66 Virus Purification and Concentration Lentivirus Purification with Vivapure® Lentiselect Kit
Vivapure® Lentiselect 40
Fast purification of up to 8 × 108 viral particles

Vivapure® Lentiselect 40 is optimally centrifuge. Additionally, this

suited for lentivirus purification for up chromatographic procedure leads to
to 40 mL cell culture and contains all pure virus samples in contrast to the
components necessary for 4 crude ultracentrifuge pellet, resulting
purifications. Up to 8 × 108 viral in higher reproducibility and increased
particles are recovered in less than gene transfer efficiency.
one hour. In contrast to traditional
ultracentrifu­gation methods, virus Typical Performance
purification with Vivapure® Lentiselect For a normal yielding vector, 2 × 15 cm
is fast and simple, without the need for culture plate purified using this
expensive equipment like an ultra­ method yield up to 8 × 108 particles.

Ordering Information and Kit Contents

Vivapure® Lentiselect 40 VS-LVPQ040

Lentiselect unit 4
50 mL syringe 4
10 mL syringe 4
Tube set with one-way valve 4
Loading buffer (10 ×) 30 mL
Washing buffer 150 mL
Elution buffer 20 mL
Vivaspin 20, 100 kDa MWCO
®
8
Instructions 1 each for Kit and Vivaspin®

Technical Data

Kit specifications
Sample size 40 mL cell culture
Number of purifications 4 × 40 mL
Infective particles (P) per mL Typically up to 3 × 109
VP | IU 5 – 15
Processing time Typically 45 minutes
Endotoxin level < 0.025 EU/mL

10 mL syringe
containing
elution buffer

Lentiselect
Unit
Lentiselect
Unit

Purified virus
Tube containing
24 mL buffer

1.  2. Washing 3. Elute

Virus Purification and Concentration Vivapure® Lentiselect 40 67

 Vivapure® Lentiselect 500
Fast purification of up to 2–5 × 109 infective particles per mL from 500 mL cell culture

Vivapure® Lentiselect 500 is optimally Vivapure® Lentiselect 500 offers

suited for VSV-G pseudotyped lenti­ a fast and simple solution for purifying
virus purification from up to 500 mL VSV-G pseudotyped lentiviruses
cell culture and contains all reagents making expensive purification
and devices necessary for purifying up equipment like ultracentrifuges
to 2–5 × 109 infective particles. redundant.

The whole purification procedure Typical Performance

is simply operated by a laboratory For a normal yielding vector, 500 mL
pump, which minimizes hands-on cell culture purified using this method
time. Unlike conventional purification yield up to 2–5 × 109 infective particles
methods as ultracentrifugation, per millilitre (total volume 1 mL).

Ordering Information and Kit Contents

Vivapure® Lentiselect 500 VS-LVPQ500

Lentiselect unit 1
Sartopore 2 150 ®
1
50 mL syringe 1
Tube set with one-way valve 1
Loading buffer (10 ×) 30 mL
Washing buffer 170 mL
Elution buffer 30 mL
Vivaspin 20, 100 kDa MWCO
®
2
Operating manual 1 each for Kit and Vivaspin®

Technical Data

Kit Specifications
Sample volume 500 mL cell culture
Number of purifications 1 × 500 mL
Infective particles (IP) per mL Typically up to 2–5 × 109*
Processing time Typically up to 3 hours
Endotoxin level < 0.025 EU/mL

1. Filter 2. Add Loading Buffer 5. Elute purified virus

3. Prepare Lentiselect Unit 6. Concentrate and exchange
4. Bind virus an d wash away contaminants buffer using Vivaspin®
* 1 mL final elution sample

68 Virus Purification and Concentration Vivapure® Lentiselect 500

 Vivapure® Lentiselect 1000
Pump driven kit for larger sample volumes

Vivapure® Lentiselect 1000 is the devices for optimal convenience. The

direct scale up kit to Lentiselect 500, traditional time consuming
for VSV-G pseudotyped lenti­virus ­ultracentrifugation method is replaced
­purification. The rapid 6 hour protocol by this fast and simple Vivapure®
results in a recovery of 4–5 × 109 ­Lentiselect 1000 kit.
­infective particles per mL (total volume
2 mL) from 1 L cell culture ­supernatant. Typical Performance
For a normal yielding vector, 1 L cell
This kit is to be operated by a culture purified using this method
laboratory pump and contains all yield up to 4 – 5 × 109 infective particles
necessary buffers and ultrafiltration per millilitre (total volume 2 mL).

Ordering Information and Kit Contents

Vivapure® Lentiselect 1000 VS-LVPQ1000

Lentiselect unit 2
Sartopore® 2 150 1
50 mL syringe 1
Tube set with one-way valve 1
Loading buffer (10 ×) 30 mL
Washing buffer 170 mL
Elution buffer 60 mL
Vivaspin 20, 100 kDa MWCO
®
2
Operating manual 1 each for Kit and Vivaspin®

Technical Data

Kit specifications
Sample volume 1 L cell culture
Number of purifications 1×1L
Infective particles (IP) per mL Typically up to 4 – 5 × 109*
Processing time Typically up to 6 hours
Endotoxin level < 0.025 EU/mL

1. Filter 2. Add Loading Buffer 5. Elute purified virus

3. Prepare Lentiselect Units 6. C
 oncentrate and exchange
4. Bind virus and wash away contaminants buffer using Vivaspin®

* 2 mL final elution sample

Virus Purification and Concentration Vivapure® Lentiselect 1000 69

70 Chapter Site Title
Application Notes
Table of Contents

1. Desalting and Buffer Exchange with Vivaspin® Centrifugal

Concentrators 72

2. Treatment of Vivaspin® Concentrators for Improved Recovery

of Low-concentrated Protein Samples 75

3. Scouting Protein Purification Conditions Using Vivapure® Centrifugal

Ion Exchange Membrane Absorbers 78

4. Concentration and Purification of Viruses by using Ultrafiltration,

Incl. Coronavirus – a Short Review 83

5. Sartorius Ultrafiltration Products in the Preparation of Biological

Nanoparticles and Medical Nanocarriers 95

6. Vivaflow® and Vivaspin® Workflow in Protein Research Laboratories 101

71
 1. Desalting and Buffer Exchange with Vivaspin®
Centrifugal Concentrators
Introduction The protein sample can then be
Vivaspin® centrifugal concentrators, concentrated again to the desired
with patented vertical membrane level, or the buffer exchange can be
technology, combine fast filtration repeated to reduce the salt
with high recovery of target proteins. concentration even further before a
This makes Vivaspin® the technology final concentration of the protein. This
of choice for desalting or buffer process is called dia­filtration. For
­exchange, avoiding lengthy dialysis proteins with a tendency to precipitate
steps. at higher concentrations, it is possible
to perform several diafiltration steps in
While proteins are retained by an sequence, with the protein
ultrafiltration membrane, salts can pass concentrated each time to only 5 or
freely through, independent of protein 10×. For example, if a precipitous
concentration or membrane MWCO. protein sample is concentrated to 5×
In consequence, the composition of then diluted back to the original
the buffer in the flow-through and volume, and this process is repeated a
­retentate is unchanged after protein further two times, this still results in a
concentration. By diluting the >99% ­reduction in salt concentration,
concentrate back to the original without over-concentrating the
volume, the salt concentration is protein.
lowered. The ­concentrate can be
diluted with water or salt-free buffer if
simple desalting is required; however,
it is also possible to dilute the
concentrate with a new buffer, thereby
exchanging the buffering substance
entirely. For example, a 10 mL protein
sample containing 500 mM salt, if
concentrated 100-fold still contains
500 mM salt. If this ­concentrate is
then diluted 100× with water or salt-
free buffer, the protein concentration
returns to the original level, while the
salt concentration is ­reduced 100× to
only 5 mM (i.e. a 99% reduction in salt
concentration).

72 Application Notes 1. Desalting and Buffer Exchange with Vivaspin®

 Methods 2. Centrifuge for the recommended
Select an appropriate MWCO for your amount of time at an appropriate
sample. For maximum recovery, select spin speed (see device operating
a MWCO ⅓ to ½ the molecular weight ­instructions).
of the molecule of interest. 3. Empty filtrate container and refill the
concentrator with an appropriate
1. Add protein sample up to the maxi- ­exchange solvent.*
mum fill volume of the concentrator 4. Centrifuge again as before.
(as stated in the device operating 5. Recover the concentrated, desalted
­instructions). If the sample volume sample from the bottom of the
is lower than the maximum device ­concentrate pocket with a pipette.
volume, it can be diluted to the
­maximum fill volume before the first
centrifugation step. This will increase
the salt removal rate.

1 2 3 4 5

Figure 1: Step-by-step method for desalting and concentration

* Filtrate volumes should be retained until the

concentrated sample has been analyzed.

Application Notes 1. Desalting and Buffer Exchange with Vivaspin® 73

 Results

Vivaspin® 20

MWCO 5 kDa 30 kDa 50 kDa 100 kDa

Cytochrome C BSA 1 BSA 1 IgG 1 mg/mL

0.25 mg/mL mg/mL mg/mL

Protein NaCL Protein NaCL Protein NaCL Protein NaCL

Recovery Removal Recovery Removal Recovery Removal Recovery Removal

Spin 1 100% 99% 97% 99% 97% 99% 90% 98%

Spin 2 96% 100% 92% 100% 93% 100% 87% 100%

Four Vivaspin® 20 devices of each brought up to 20 mL with ultrapure

MWCO were tested with 20 mL sam- water from an Arium® system (Sartorius).
ples. Each sample contained 500 mM OD readings were taken at 410 nm for
NaCl. To perform diafiltration, devices Cytochrome C or 280 nm for BSA and
were centrifuged at 4,000 g for 45 min IgG samples. Salt concentrations were
(5 kDa MWCO) or 30 min (>5 kDa measured using a Qcond 2200
MWCOs). After the first and second ­conductivity measuring instrument.
spins, the retentate samples were

Vivaspin® 6

MWCO 5 kDa 30 kDa 50 kDa 100 kDa

Cytochrome C BSA 1 BSA 1 IgG 1 mg/mL

0.25 mg/mL mg/mL mg/mL

Protein NaCL Protein NaCL Protein NaCL Protein NaCL

Recovery Removal Recovery Removal Recovery Removal Recovery Removal

Spin 1 98% 99% 92% 99% 93% 99% 92% 98%

Spin 2 85% 100% 86% 100% 83% 100% 89% 100%

Four Vivaspin® 6 devices of each were brought up to 6 mL with

MWCO were tested with 6 mL ultrapure water from an Arium® system
samples. Each sample contained 500 (Sartorius). OD readings were taken at
mM NaCl. To perform diafiltration, 410 nm for Cytochrome C or 280 nm
devices were centrifuged at 4,000 g for BSA and IgG samples. Salt
for 45 min (5 kDa MWCO) or 30 min concentrations were measured using a
(>5 kDa MWCOs). After the first and Qcond 2200 ­conductivity measuring
second spins, the retentate samples instrument.

Conclusions r­ emoval to 99% with >92% recovery of

As the results show, the efficient de- the target protein. Diafiltration using
sign of Vivaspin® devices allowed >95% ultrafiltration devices such as Vivaspin®
of the salt to be removed during the 6 and 20 represents a faster and more
first centrifugation step. Only one efficient solution to desalting and
­subsequent centrifugation step was ­buffer exchange, than conventional
needed to increase the typical salt techniques such as dialysis.

74 Application Notes 1. Desalting and Buffer Exchange with Vivaspin®

2. Treatment of Vivaspin® Concentrators for Improved
Recovery of Low-Concentrated Protein Samples
Introduction Table 1: Passivation Solutions
With appropriate device size and
membrane cut-off selected, Vivaspin® Type Concentration
products will typically yield recoveries Powdered milk 1% in Arium® water
for the concentrated sample > 90%
BSA 1% in PBS
when the starting sample contains
over 0.1 mg/mL protein of interest. Tween 20 5% in Arium® water
­Depending on sample characteristics SDS 5% in Arium® water
relative to the membrane type used, Triton X-100 5% in Arium® water
solute (protein) adsorption on the
PEG 3000 5% in Arium® water
membrane surface is typically very
low (2 – 10 μg/cm²) and in practice
not detectable. Passivation procedure for Vivaspin®
ultrafiltration concentrators
This can increase to 20 – 100 μg/cm²
when the filtrate is of interest and the A) Passivation procedure
sample must pass through the whole 1. Wash the concentrators once by
internal structure of the membrane. ­filling with Arium® water and spin
Whilst the relative adsorption to the the liquid through according to the
plastic of the sample container will be respective protocol.
proportionately less important than on 2. Remove residual water thoroughly
the membrane, due to the higher total by pipetting.
surface area, this can be also be a Caution: Take care not to damage
source of yield loss. Typically, a higher the membrane with the pipette tip.
cut-off membrane will bind more than 3. Fill concentrators with the blocking
a low molecular weight alternative. solution of choice as given in Table 1.
4. Incubate the filled concentrators at
Whenever possible, the smallest room temperature for at least 2
MWCO and device size applicable hours (overnight is also possible
should be chosen. Swinging bucket except for Triton X-100 which is not
rotors are preferred to fixed angle recommended for overnight
­rotors. This reduces the surface area of incubation).
the concentrator that will be exposed 5. Pour out the blocking solution.
to the solution during centrifugation. 6. Rinse the device 3 – 4 × very
thoroughly with Arium® water and
An important factor not to be finally spin through.
neglected is the thorough recovery of 7. The “passivated” devices are now
the ­retentate. Make sure to carefully ready for use. We recommend
­remove all traces of solution from the comparing different passivation
sample container and, if feasible, rinse reagents with an untreated device.
the device after recovering the sample
with one or more drops of buffer and Note
then recover again. It is necessary to rinse the device
­thoroughly before each washspin
The intention of the following “passiva- to ensure that traces of passivation
tion” procedure is to improve recovery compound are removed from the
of protein samples in the nano- to deadstop. Use the device immediately
­microgram concentration range by for protein concentration or store it at
pretreating the device (membrane & 4°C filled with Arium® water, to prevent
plastic). For this purpose a range of the membrane from drying.
solutions are suggested in Table 1.

Application Note 2. Passivation of Vivaspin® Concentrators 75

 B) Evaluation of passivation effects Results and discussion
(exemplary with BSA) As an example, the effect of milk
1. Prepare a 10 μg/mL BSA stock ­powder was analysed. It could be
­solution e.g. by diluting 90 μL of the shown (Figure 1) that the protein
4 mg/mL stock solution in 36 ml 0.1 ­recovery of a 10 μg/mL BSA solution
M sodium borate pH 9.3. Mix well. could be increased from around 70 to
2. Fill Vivaspin® 2 devices with 2 ml 90%. If milk powder is not interfering
of this 10 μg/mL BSA solution and with sample purity and quality, it is a
close with cap provided. good starting point to improve
3. Spin the device in a swing-out rotor ­recovery of diluted sample solutions.
at 4,000 × g until the volume is to
app. 100 μL. Protein recovery (10 μg/mL BSA) with
4. R  ecover the concentrate and make Vivaspin® PES 10 kDa after passivation
back up to 2 mL with 0.1 M sodium In another example, detergents were
borate pH 9.3 analysed with only 250 and 500 ng
5. Determine recovered protein BSA (Figure 2) BSA recovery declined
­concentrations e.g. according to to 50 – 30% in untreated devices as
Bradford or BCA assays. the protein concentration was
reduced. Significant improvement to
60 – 90% recovery could be
demonstrated when using the
passivation strategy. Often, Triton
X-100 seemed to work though the
optimal reagent has to be selected for
the respective protein and its
hydrophilic | -phobic characteristics.

76 Application Note 2. Passivation of Vivaspin® Concentrators

 Recovery comparison of passivation with milk powder

100

80

60

40

20

0
milk powder untreated

Figure 1: Protein recovery (10 µg/mL BSA) with Vivaspin® PES 10 kDa after passivation

Recovery comparison of passivation with common buffer solutions

PEG Triton X-100

100 Tween untreated

80

60

40

20

0
250 ng 500 ng

Figure 2: Protein recovery (250 and 500 ng BSA) with Vivaspin® 2 PES 10 kDa after passivation

Summary device will reduce losses on the plastic

Passivation is an appropriate method surface. One very important thing to
to achieve increasing sample recovery remember is that the blocking agent is
when using very dilute samples. In ad- potentially introduced into the sample.
dition to skimmed milk, other proteins It should be assured that this will not
(BSA), detergents and compounds are interfere with downstream analysis.
possible. However, it should be noted
that this is a general procedure, not For example, proteins must not be
specific for any particular application. used for passivation if a pure protein
Depending on the hydrophilic | is intended to be concentrated for
­-phobic character of the protein x-ray crystallography, as even the
non-specific binding may be more or smallest traces would interfere with
less of a problem and the suggested the diffraction pattern. Other subse-
passivation solutions may lead to dif- quent analyses methods include
ferent results. Even with the Hydrosart ­activity testing, gel electrophoresis
membrane, which is recommended or labelling are less problematic.
for dilute samples, passivation of the

Application Note 2. Passivation of Vivaspin® Concentrators 77

 3. Scouting Protein Purification Conditions Using
Vivapure® Centrifugal Ion Exchange Membrane Absorbers
Introduction The results from this experiment
For separation and purification of provided the optimal binding pH and
­proteins from biological samples, the best ion exchange chemistry for
­different characteristics of the target the purification of SH2 domain.
protein e.g. its size, charge,
hydrophobicity or specifically In a second step, the best elution
engineered tags, are exploited. method was evaluated by applying
­increasing salt concentrations to
With ion exchange chromatography, ­columns which were shown to bind
separation is achieved on the basis of the target protein in step one, leading
charge differences between to a complete purification protocol in
biomolecules. This makes it a versatile less than one hour.
method often used for pre-
fractionation or ­purification of a target Materials and Methods
protein from crude protein mixtures. Buffers tested to determine the
To optimize the purification procedure optimum pH and salt concentration
for an i­ndividual target, several binding for binding and elution in ion
and elution conditions have to be exchange purification of SH2 domain
tested on cation and anion exchange
­matrices. Buffer A: 25 mM Citrate, pH 4

Buffer B: 25 mM Potassium phosphate, pH 6

In contrast to traditional column
Buffer C: 25 mM HEPES, pH 8
­chromatography methods, Vivapure®
IEX centrifugal columns allow scouting Buffer D: 25 mM Sodium bicarbonate, pH 10
of several chromatography conditions Buffer E: 25 mM Citrate, pH 4, supplemented
in parallel, leading quickly to different with 1 M NaCl.
fractions which can be further analyzed Buffer F: 25 mM Potassium phosphate, pH 6,
for enriched or even already purified supplemented with 0.2 M, 0.4 mM,
target protein. 0.6 mM, 0.8 mM and 1 M NaCl,
­respectively.

Here, we demonstrate the performance Buffer G: 25 mM HEPES, pH 8, supplemented

with 1 M NaCl
of Vivapure® IEX Mini spin columns for
evaluation of optimal purification Buffer H: 25 mM Sodium bicarbonate, pH 10,
supplemented with 1 M NaCl
conditions of cloned SH2 domains
from an E. coli lysate in a two step
procedure. This protocol can generally
be employed for identifying a
purification method based on ion
exchange chromatography for a given
target protein, as it is fast and only uses
small amounts of the sample.

In the first step of this study, binding

conditions were evaluated by loading
the sample on Vivapure® Q and S
­columns at various pH values, eluting
bound proteins with a high salt
concentration buffer and analyzing all
fractions for the target protein.

78 Application Note 3. Scouting Protein Purification Conditions

Scouting Binding Conditions Afterwards, Vivapure® Mini spin
300 mL LB media was inoculated ­columns were reloaded with 400 μL
with 4 mL of an overnight culture and sample and spun again for 5 min at
incubated at 37°C, shaking at 150 rpm 2,000 g. Loosely bound proteins were
until an OD600 of 1.0 was reached. washed away with the application of
IPTG was added to a final 400 μL of the respective binding
concentration of 1 mM and the culture buffer to each of the columns and
incubated for a further 4 h with shaking ­spinning for 5 min at 2,000 g. ­Flow-
at 150 rpm. Cells were harvested by through and wash fractions were
centrifugation at 4,000 g for 30 min at collected for subsequent ­detection of
4°C. The pellet was resuspended in 35 the target protein.
mL PBS (150 mM KPi, pH 7.3) and cells
were lyzed by addition of lysozyme to a 200 μL of elution buffer E, F, G or H,
final ­concentration of 0.1 mg/mL and were applied to the washed columns
­incubation for 1 h at 37°C. Insoluble and spun for 3 min at 2,000 g. Eluates
particles and cell debris were removed were saved for subsequent analysis.
by centrifugation at 10,000 g for
30 min at 4°C. 4 μL of flow-through, wash, and eluate
fractions from each column were
4 × 200 μL aliquots of the cell lysate analyzed by reducing SDS-PAGE
were diluted with 1.8 mL binding buffer followed by silver staining.
A to D, to adjust each sample to the
­respective pH being tested. To avoid Optimizing Elution Conditions
clogging of the membranes in the Taking account of the results of the
­Vivapure® Mini spin columns, samples first experiment (Scouting Binding
were clarified by passage through Conditions) 200 μL cell lysate was
0.45 μm CA Minisart NML syringe ­diluted with 1.8 mL binding buffer B
­filters (Sartorius). (25 mM KPi, pH 6). To avoid clogging
of the membrane in the Vivapure® Mini
4 × Q and 4 × S Vivapure® Mini spin spin column, the pH adjusted sample
columns were labeled 4, 6, 8 and 10, was clarified by passage through a
corresponding to the pH of the buffer 0.45 μm CA Minisart NML syringe
to be used. To each spin column, ­filter (Sartorius).
400 μL of the corresponding binding
buffer was added and spun for 5 400 μL binding buffer B was applied
minutes at 2,000 g (45° fixed angle to one Vivapure® S Mini spin column
rotor). and spun for 5 minutes at 2,000 g.

400 μL of the clarified samples 400 μL of the clarified sample was

adjusted to pH 4, 6, 8 or 10 were ­applied to the equilibrated Vivapure® S
applied to each of the correspondingly column and spun for 5 min at 2,000 g.
equilibrated Vivapure® Q and S spin Afterwards, the Vivapure® S Mini spin
columns. ­Columns were spun for 5 min column was reloaded with 400 μL
at 2,000 g. sample and spun again for 5 min at
2,000 g.

Application Note 3. Scouting Protein Purification Conditions 79

 Loosely bound proteins were washed Results
away by application of 400 μL binding Dilution of the E. coli lysate with
buffer to the column and spinning for binding buffer A (25 mM Citrate, pH 4)
5 min at 2,000 g. Flow-through and lead to complete precipitation of
wash fractions were saved sample proteins. Thus, pH 4 could not
for analysis. be tested in this experiment. As can be
seen on the SDS gel in (Figure 1), the
To elute the target protein, 100 μL target protein was present in the
­elution buffer F, supplemented with eluates from Vivapure® Q Mini spin
0.2 M NaCl was applied to the columns at all pH values tested,
­Vivapure® S Mini spin column and together with most of the E. coli
spun for 3 min at 2,000 g. The eluate proteins (Lanes Q “e”). In contrast,
was collected. For the next elution using the Vivapure® S Mini spin
step, 100 μL of elution buffer F, column, at all pH values tested, most E.
­supplemented with 0.4 M NaCl was coli proteins did not bind to the
applied and again spun for 3 min at membrane and were found in the
2,000 g. Elution was continued with flow-through (Lane S “f”), thus
0.2 M NaCl increments until a final resulting in purer target protein in all
salt concentration of 1 M was reached, eluate fractions (Lane S “e”).
saving the eluates from each step.
Differences could be detected in
4 μL of flow-through, wash, and eluate the binding efficiency of the target
fractions from each column were protein. At pH 8, traces of the target
analyzed by reducing SDS-PAGE protein were already found in the flow-
followed by silver staining. through, with slightly higher amounts
at pH 10 (Lane S “e”). At pH 6, the most
efficient binding of the target protein
to the S membrane was observed.

The purification conditions

determined for Vivapure® S with
potassium phosphate buffer (pH 6)
were further optimized to determine
the ideal salt concentration for SH2
domain elution. The target protein
started to elute with 200 mM NaCl,
however the main ­fraction eluted with
400 mM NaCl. Traces of the target
protein were also found in the next
elution step with 600 mM NaCl, but
this might be due to the low elution
volume.

80 Application Note 3. Scouting Protein Purification Conditions

Figure 1

pH = 6 pH = 8 pH = 10

66 kDa

45 kDa

Target
protein
31 kDa

22 kDa

Sample Sample Volume (μL) Volume Loaded Onto Gel (μL)

M = Broad range marker

s = Sample before application 800 4

f = Flow-through 800 4

w = Wash fraction 400 4

e = Elution with 1 M NaCl 200 4

Note: Scouting for optimal binding conditions of a SH2 domain expressed in E. coli. 12% reducing
SDS gel, silver stained, shows the sample before purification (s), flow-through (f), wash (w) and
­eluate (e) fractions (1 M NaCl) from Vivapure® Q and S Mini spin columns, at the various pH values
tested.

Application Note 3. Scouting Protein Purification Conditions 81

 Figure 2

66 kDa

45 kDa

Target
protein
31 kDa

22 kDa

Sample Process Volume (µL) Volume Loaded Onto Gel (µL)

M = Broad range marker

s = Sample before application 800 16

f = Flow-through 800 16

w = Wash fraction 400 16

e1 = 25 mM KPi, pH 6, 200 mM NaCl 100 8

e2 = 25 mM KPi, pH 6, 400 mM NaCl 100 8

e3 = 25 mM KPi, pH 6, 600 mM NaCl 100 8

e4 = 25 mM KPi, pH 6, 800 mM NaCl 100 8

e5 = 25 mM KPi, pH 6, 1 M NaCl 100 8

Note: Optimizing elution conditions for a SH2 domain expressed in E. coli, using Vivapure® S Mini
spin column at pH 6. 12% reducing SDS gel, silver stained, shows the sample before purification (s),
flow through (f), wash (w) and eluate (e1-5) fractions.

-
Conclusion The results obtained in this experiment
A two-step procedure was used to can be used to various ends, e.g:

--
rapidly scout optimal purification Polishing a specific protein after
conditions for a target protein (a SH2 ­purification with another chromato-
domain from E. coli lysate) with ion graphic technique
exchange chromatography. In the first Quickly establishing a FPLC method
step, the most suitable ion exchanger for a new protein
and buffer pH for binding the target Identification of the optimal purifica-
protein was verified. In the second step, tion method prior to scale up with
the elution condition was optimized, ­Vivapure® IEX Maxi spin columns.
building on the results gained in step
one. With the scouting procedure For these purposes Vivapure® IEX Mini
described here, it was possible to and Maxi spin columns and Sartobind®
quickly and conveniently purify the membrane adsorber units with FPLC
target protein to homogeneity. connectors are available.

82 Application Notes 3. Scouting Protein Purification Conditions

4. Concentration and Purification of Viruses by Using
Ultrafiltration, Incl. Coronavirus – A Short Review
Introduction This short review highlights methods
Evolutionary, viruses developed ­various for the purification of various
mechanisms to interact and ­manipulate mammalian viruses for basic medical
the genetic material of their target cells. research. Also, the concentration of
Based on this, m ­ odern molecular pathogenic viruses from water and
­biology utilizes ­viruses in a constantly food samples and the purification of
growing number of ­applications.¹ marine bacteriophages (virioplankton)
They range from controlled genetic are highlighted. It will also give
transfection of cells to a variety of guidance for the selection of an ideal
different basic studies in medical performing device with the optimum
science.² In medical studies the molecular weight cut-off (MWCO) for
strategic focus is on ­recombinant the user specified ultrafiltration
vaccines and on the ­development of process.
potential vectors for gene ­therapy.³,⁴
Concentration of mammalian viruses
Besides the great relevance of viruses in medical research
for medical applications, the In medical research viruses and VLPs
assessment of virus type and content are of major interest, particularly for
is important for the risk assessment of ­investigations on infectious viral
food and drinking water.⁵ Also, the ­diseases and for the development of
classification of virus content is often vaccines or antiviral drugs. Moreover,
of high ­relevance for the quality certain VLPs can manipulate genetic
control of aquatic biotopes.⁶ material in a directed manner and are
used broadly in the development of
During the preparation, handling, or genetic therapy approaches.
analysis of viruses orvirus-like particles Additionally, viral vectors are well
(VLPs), a concentration and | or established as a transfection method
purification step is frequently required.⁵ for gene transfer to cell lines e. g. to
­Typical viruses have a size within the manipulate mammalian cells in vivo
range of about 20 nm up to several and in vitro.
hundred nanometers.⁷ Therefore they
are ideally suited for the retention on An overview of thematically linked
ultrafiltration membrane systems and publications using Sartorius
such ultrafilters are widely used in basic ultrafiltration devices for the
virus research. The specifications of purification and concentration of
such ultrafiltration devices depend on viruses and VLPs in the medical
the particular type of virus and the context is given in Table 1. Among
­purpose of the subsequent ­application. other applications, Vivaspin® devices
were employed for the concentration
of adeno-associated virus (AAV) and
lentiviral vectors after ­purification via
ion exchange chromatography,⁸–¹⁰ on
blood sera to ­prepare blank samples
from hepatitis C virus (HCV)-positive
blood sera,¹¹ for the ­development of a
vaccine against human
immunodeficiency virus (HIV) and of
an antiviral drug against C ­ hikungunya
virus.¹²,¹³

Application Notes 4. Concentration and Purification of Viruses 83

 Table 1: Summarized examples of applications using Vivaspin® and Vivaflow®
for viruses in medical research

Goal of Research Purpose of Filtration Sartorius Subsequent Step Ref.

(Type of virus, (Buffer System) ­Ultrafiltration
Host Organism) Device (MWCO)

Gene therapy Diafiltration Vivaflow® Storage, chromatography 14

(Adenovirus type 5, (20 mM Tris saline (100 kDa) on Sartobind® STIC
VLP, human) buffer) ­membrane absorber
(FPLC)

Reduction of Removal of HCV Vivaspin® Preparation of negative 11

­HCV-induced from human blood (30 kDa) control (from positive
fibrosis (Hepatitis C serum (Blood serum) ­sample) for immunofluores-
Virus; human) cence assay, fibrosis
­induction assays

Development of a Removal of protein Vivaspin® 20 Virus inactivation 12

viral entry inhibitor for fraction from virus (1,000 kDa)
HIV (HIV, human) (PBS)

Gene therapy for Concentration and Vivaspin® 20 Titer, ELISA, cell binding 8
­cancer treatment ­purification after (1,000 kDa) assay, apoptosis | cell cycle
­(adeno-associated ­expression, Buffer assay
virus; rAAV-2, human) ­exchange after
His tag (FreeStyle 293
Expression Medium
(Gibco), serum-free)

System for controlled Concentration of Vivaspin® 20 Transduction of mice 9

gene expression in eluate after anion (100 kDa) ­neurons
mice brain (Adeno-­ ­exchange chromatog-
associated virus, mice) raphy (elution buffer)

Efficient gene transfer Concentration after Vivaspin® Quantification via real- 10

into the CNS ion exchange (100 kDa) time PCR and end-point
(Lentivirus, human) ­chromatography ­dilution. Transduction of
(PBS) murine neuronal and glial
cells in vivo

Identification of Concentration Vivaspin® 20 Quantification by TCID₅₀ 13

­effective chikungunya (100 kDa)
antiviral drugs
(Chikungunya-Virus,
human)

Gene therapy of Concentration Vivaspin® 4 Titer determination 15

­achromatopsia in (Anion exchange (10 kDa) by ­dot-blot analysis,
mice (Recombinant ­chromatography subretinal injections
­adeno-associated elution buffer)
virus, human virus
used in mice)

84 Application Notes 4. Concentration and Purification of Viruses

Concentration of viruses from independent of the chemical
drinking water and food samples properties and the structure of the
The guidelines for drinking-water virus.¹⁷ Thus ultrafiltration is very well
­quality by the world health suited to isolate and concentrate virus
organization describe safety plans to particles from water samples and is a
reduce potential risks from various valuable aid during the assessment of
virus i­nfections.¹⁶ It states that, due to water quality. Most of the viruses
the increased resistance of viruses to which are found in water and also food
­disinfection methods, an absence of samples are of fecal origin. Screening
bacterial contamination after for these viruses is crucial to prevent
disinfection cannot be used as a infections. The most frequent ones are
reliable ­indicator of the presence | hepatitis A, hepatitis E and norovirus.¹⁸
absence ­of pathogenic viral species in Ultrafiltration has been described as
drinking water supplies. Considering the most appropriate method for the
this, ­ultrafiltration can play a vital role recovery of hepatitis A virus from
in ­detecting such viral contaminations vegetables and other food items.¹⁹
for the research on drinking water Detection of infectious viruses is
quality and food safety. mainly done by propagation in cell
culture (plaque assay) or the ­detection
For an ultrafiltration step, the water of the viral genomes by ­molecular
sample does not have to be pre- amplification techniques such as
conditioned and its efficacy in quantitative reverse transcriptase
concentrating the virus is virtually polymerase chain reaction (RT-PCR).²⁰

Table 2: Summarized examples of ultrafiltration applications using Vivaspin®

and ­Vivaflow® for viruses from drinking water and food samples

Goal of Research Purpose of Filtration Sartorius Ultra- Subsequent Step Ref.

(Type of virus, (Buffer System) filtration Device
Host Organism) (MWCO)

Method for the Concentration (PBS Vivaspin® RNA extraction for 22

­detection of norovirus processed (5 kDa) real-time RT-PCR
genogroup I food samples)
­(Norovirus, human)

Analysis of viral con- Concentration of Vivaflow® 200 Qualitative analysis 21

tent in groundwater drinking water sample (10 kDa) (enterovirus) by
(A set of pathogenic (Drinking water) ­RT-nested PCR and
viruses, potentially microtiter neutralization
human) test

Comparative Analysis Concentration Vivaspin® 20 RNA extraction for 19

of Viral Concentration (0.25 M threonine, (100 kDa) real-time RT-PCR
Methods (Hepatitis A 0.3 M NaCl, pH 9.5)
virus, human)

Analysis of regional Concentration Vivaspin® 2 Nucleic acid extraction 23

outbreak of gastro- (50 mmol/L glycine
enteritis due to drink- buffer, 1% beef
ing water contamina- extract)
tion (Norovirus,
Astrovirus, Rotavirus,
Enterovirus, Hepatitis
A virus; human)

Application Notes 4. Concentration and Purification of Viruses 85

 Concentration of viruses and chemical and structural properties
bacteriophages from marine of the viruses. Thus, it finds wide use
biological samples for the analysis of aquatic viruses.
In marine biology, the concentration For instance, Schroeder et al. (ref. 26)
and subsequent analysis of marine were able to determine the diversity
bacteriophages (virioplankton) is of and monitor population dynamics of
major interest. They outnumber the viruses that infect Emiliania huxleyi, a
bacterioplankton (their host globally important form of
organisms) by an order of magnitude photosynthetic plankton. In this study
and thus have an important influence a reusable Vivaflow® 50 unit equipped
on the whole marine biosphere.²⁴ with a polyethersulfone (PES)
membrane with MWCO of 50 kDa was
As described by Wyn-Jones & used to concentrate viruses in sea
­Sellwood (ref. 17) ultrafiltration can water s­ amples prior to storage and
be used to concentrate virus particles analysis. For further examples of virus
in water samples without any prior concentration from marine biological
­pretreatment of the sample and it is samples see table 3.
also practically independent from the

Table 3: Summarized examples of ultrafiltration applications using Sartorius

­Vivaflow® and Vivaspin® for samples from marine biology

Goal of Research Purpose of Filtration Sartorius Ultra- Subsequent Step Ref.

(Type of virus, (Buffer System) filtration Device
Host Organism) (MWCO)

Assessment of 0.2 μm filtration for Vivaflow® 200 Subsequent analysis 25

virioplankton diversity clarification, filtrate (0.2 μm and by DNA separation
(Virioplankton, Plankton) subjected to 3 kDa 30 kDa) on Agarose gel
filter for concentration
(Sea water)

Classification of virus Vivaflow® 200: harvest Vivaflow® 200, Classification of 28

(MpRNAV-01B, and concentration Vivaspin® new virus: genome,
Micromonas pusilla) of whole cell lysate; (30 kDa) proteins, stability,
Vivaspin®: washing etc.
(removal of CsCl)

Assessment of genetic After 0.45 μm filtration, Vivaflow® 50 PCR and Denaturing 26

diversity in virioplankton concentration 1 L to (50 kDa) gradient gel
(Emiliania huxleyi Bloom 20 mL (Sea water) electrophoresis
virus, Eukaryotic
­phytoplankton - alga)

Investigation of gene Concentration Vivaflow® 50 CsCl-gradient 27

­expression during from 5 L to 20 mL (50 kDa)
infection (Emiliania (f/2 medium)
­huxleyi virus strain
86, Eukaryotic
­phytoplankton - alga)

Study on host genome Clarification with 0.2 μm Vivaflow® 200 CsCl gradients, 29
integration (virophage filter and concentration (0.2 μm and electron microscopy
mavirus, Cafeteria with 100 kDa filter 100 kDa)
roenbergensis) (Cafeteria roenbergen-
sis, f/2 medium)

86 Application Notes 4. Concentration and Purification of Viruses

Concentration of Coronavirus A key component to the infection
for general research and protein cycle is the coronavirus spike (S)
­research (spike protein) ­protein, that mediates entry into host
Coronaviruses are spherical, enveloped, cells, through both attachment and
RNA based viruses that are typically membrane fusion. As such, it is a
80 – 120 nm in diameter, but in many primary target for the development of
cases have a diameter outside of this novel antiviral drugs and vaccines.
range. Coronavirus genomes are the
largest of all RNA viruses which ­offers The concentration and purification of
a relatively large area of study. both the virons and the spike proteins
Correlatingly the potential for future from cell culture and supernatants is
mutations in this large genome may often a key requirement to isolate the
lead to future human diseases that respective target, prior to structural,
may evolve into epidemics and functional analysis and binding assays,
pandemics, such as the previous etc.
Middle East Respiratory Syndrome
(MERS-CoV), and Severe Acute Table 4 highlights several applications
Respiratory Syndrome 1 (SARS-CoV-1) where Vivaspin® centrifugal
and 2 (SARS-CoV-2). Hence further concentrators, or Vivaflow® tangential
research into the replication, flow f­ iltration cassettes have been
transmission, genome and structure used for the concentration or
will continue with greater investment Coronavirus proteins, including the
of time and funding in the years to spike protein. References are also
come. provided to direct readers for detailed
reading.

Table 5 provides examples of

concentration of intact virons, or
Coronavirus virus like particles (VLPs),
with the same devices.

Application Notes 4. Concentration and Purification of Viruses 87

 Table 4: Summarized examples of ultrafiltration applications using Sartorius
­Vivaflow® and Vivaspin® for coronavirus protein samples.

Goal of Research Purpose of Filtration Sartorius Subsequent Step Ref.

(Type of virus, Host (Buffer System) Ultrafiltration
Organism) Device
(MWCO)

Neutralization of a Buffer exchange of a Vivaspin® 20, Protein concentra- 30

­SARS-CoV-2 antibody to SARS-CoV-2 RBD PES (10 kDa) tion by UV/Vis and
a functionally conserved ­protein binding affinity
receptor binding domain by Streptavidin BLI
(RBD) on the trimeric (Sartorius Octet)
spike (S) protein

Investigation of Concentration of Vivaspin® 20, Purification by SEC 31

­neutralising antibody ­SpyTag-RBD protein PES (10 kDa)
­response on a SARS- construct
CoV-2 spike glycoprotein
RBD-SpyVLP (virus-like
particle) platform

Investigation of exosome Concentration of Vivaspin®, PES Western blot 32

based vaccines contain- ­solubilized spike protein (10 kDa) ­analysis
ing coronavirus spike (S) in supernatant
protein, for SARS-CoV-1

Analyze of the ability to Concentration of Vivaspin®, PES Western blot 33

redirect the functionality cellular receptor protein ­analysis
of the Mouse Hepatitis ­constructs
coronavirus spike (S) pro-
tein to infect
human cancer cells

Structure determination Concentration of Vivaspin®, PES Crystalization 34

of coronavirus SARS- ­coronavirus nsp1 during screening
CoV-1 non-structural purification process
­protein 1 (nsp1)

Structure determination Concentration of Vivaspin®, PES Crystalization 35

of the ADRP domain ­coronavirus nsp3 during (10 kDa) screening
of Feline Coronavirus purification process
(FCoV) non-structural
protein 3 (nsp3)

Investigation into the role Concentration of VLPSs Vivaspin®, PES Cell-cell fusion 36
of three transmembrane from HEK 293T cell assay
proteases in the activa- ­culture supernatant
tion of SARS-CoV-1 spike
(S) protein

Cryo-electron micros­ Concentration of Vivaflow®, PES Affinity purification 37

copy of Human Corona­ ­recombinant HCoV- (10 kDa)
virus HCoV-NL63 spike NL63 viruses from
glycoprotein trimer that ­clarified Drosophila S2
is a potetial target for cell culture supernatant
neutralizing antibodies
during infection

88 Application Notes 4. Concentration and Purification of Viruses

Table 5: Summarized examples of ultrafiltration applications using Sartorius
Vivaspin® for coronavirus viron and VLP samples

Goal of Research Purpose of Filtration Sartorius Subsequent Step Ref.

(Type of virus, Host (Buffer System) Ultrafiltration
Organism) Device
(MWCO)

Characterisation of Concentration of Vivaspin®, PES Quantification 38

­phenotypic changes in MERS-CoV virus isolates (100 kDa) using plaque titra-
virus isolates, such as tion Viral RNA
MERS-CoV, that could ­sequencing analysis
relate to pandemic
­potential

Investigation of ­antiviral Concentration of MERS- Vivaspin® 20, Limiting ­dilution 39

potential of Echinacea CoV and ­SARS-CoV virus PES assay (TCID₅₀)
purpurea (Echinaforce®) dilutions
against human
­coronaviruses; SARS-
CoV and MERS-CoV

Investigation into Concentration and Vivaspin® 500, Inoculation 40

­inactivation of ­ ­separation of deactivat- PES onto Vero-E6
SARS-CoV-2 through ed ­SARS-CoV-2 from monolayer
heating and chemical lysis buffer
protocols

Investigation of viral and Concentration of SARS- Vivaspin®, PES Western blot 41,
cellular determinants CoV and hCoV-EMC ­analysis 42
governing hCoV-EMC virus like particles (VLPs)
entry into host cells

Application Notes 4. Concentration and Purification of Viruses 89

 Concentration and capture of virons The severity of this pandemic is driving
and | or viral RNA in wastewater increased research and funding in all
In humans and birds Coronaviruses associated areas. Once area is on the
may inflict mild to fatal respiratory tract tracking and epidemiological studies
infections, but in other animal groups of SARS-CoV-2 infections. One area
a range of other diseases may also of focus is in the use of regional
occur, such as hepatitis and wastewater systems, where the
neurological illness⁴². SARS-CoV-2 is compartmentalisation of these
the most recent among a string of systems offers distinct tracking in real
Coronavirus epidemics, which early time, without the lag for symptom
indications suggest that due to its high appearance and clinical diagnosis⁴³. In
infectivity, rates of asymptomatic addition, the data collected can be
infection, ­significant incubation time, used as a supplemental and low-cost
our relatively limited knowledge of surveillance indicator on the
transmission dynamics and overall lack circulation of the virus in a community
of g
­ lobal pandemic preparation, has without the need to screen individuals.
evolved into a true global pandemic Further, it c
­ ontributes to the tracking
and has caused significant impact on of infection prevalence, by adding
global health, society and economy. another epidemic indicator⁴⁴.

RT-PCR is the standard method to

test for SARS-CoV-2, but samples
­typically require concentration and
­removal of non-Coronavirus material
prior to testing to ensure optimal
­results. Ultrafiltration is a successful
method for this⁴³, and some examples
have been given in Table 6.

90 Application Notes 4. Concentration and Purification of Viruses

Table 6: Summarized examples of ultrafiltration applications using Sartorius
­Vivaflow® and Vivaspin® for virus and viral RNA in wastewater samples

Goal of Research Purpose of Filtration Sartorius Subsequent Step Ref.

(Type of virus, Host (Buffer System) Ultrafiltration
Organism) Device (MWCO)

Measurement of SARS- Concentration of viral Vivaspin®, PES Viral RNA extraction 43,
CoV-2 RNA in sewage RNA (50 kDa) and purification RT- 44,
qPCR quantification 45

Benchmarking virus Concentration of viral Vivaspin®100, Viral RNA extraction 46

concentration methods RNA PES (10 kDa) and purification RT-
for quantification of SARS- qPCR quantification
CoV-2 in raw wastewater

Evaluation of two Concentration of viral Vivaspin® Viral RNA extraction 47

methods to concentrate RNA from 40 ml (total) (10 kDa) and RT-qPCR and
SARS-CoV-2 from to 700-1000 µl ddPCR
untreated wastewater quantification

Virus detection in full scale Concentration of viral Vivaflow® 50, PEG precipitation 48
membrane bioreactor particles in effluent PES Viral
(MBR) plant by virus RNA quantification
concentration monitoring,
inc. Norovirus, Sapovirus
and Rotavirus

Evaluation of membrane Concentration of Vivaflow® 50, Nucleic acid 49

bioreactor wastewater effluent from 1 L to 40 PES extraction
virus removal, inc. mL RT-PCR
Norovirus, Sapovirus, quantification
Adenovirus

Evaluation of membrane Concentration of Vivaflow® 50, Nucleic acid 50

bioreactor wastewater viruses in effluent PES extraction
Norovirus removal RT-PCR
quantification

Evaluation of the Concentration of Vivaflow® 50, Nucleic acid 51

association between viruses in effluent PES extraction
number of hepatitis E RT-qPCR
cases in the community quantification
and concentration in local
sewage

Application Notes 4. Concentration and Purification of Viruses 91

 Conclusion Abbreviations
The purification of virus by ultrafiltration AAV Adeno-associated virus
is virtually independent of the chemical CNS Central nervous system
properties and the structure of the virus DNA Deoxyribonucleic acid
particles. As viruses have a size within CoV Coronavirus
the range of about 20 nm up to several ELISA Enzyme-linked
hundred nanometers, they are typically ­immunosorbent assay
several orders of magnitude bigger FPLC Fast protein liquid
than even the largest protein ­chromatography
complexes.⁷ Therefore, most viruses are fCoV Feline Coronavirus
unfailingly retained on membranes with hCoV Human Coronavirus
MWCOs up to 1,000 kDa. The exact HCV Hepatitis C virus
specifications of the ideal ultrafiltration HIV Human immunodeficiency
membranes depend on the purpose of virus
the subsequent application. kDa Kilodalton (1,000 g per
mole)
Ultrafiltration for the concentration of M Molarity (mole per litre)
Coronavirus species plays and MERS Middle east respiratory
important role in a range of workflows. ­syndrome
­Perhaps due to the size distribution of mol Mole
viruses and VLPs, the exact MWCO MWCO Molecular weight cut-off
used is not standard across each study. nsp Nonstructural protein
Although typically, for 80 – 120 nm PBS Phosphate buffered saline
­particles the 100 kDa MWCO would PCR Polymerase chain reaction
provide the optimal balance between PEG Polyethylene Glycol
recovery, removal of interfering PES Polyethersulfone
­substances, speed and shear stresses. RNA Ribonucleic acid
Whereas for the recovery of RNA SARS Severe acute respiratory
­material, lower MWCOs (10 – 50 kDa) ­syndrome
are recommended to capture a greater RBD Receptor binding domain
range of RNA chain lengths. However, BLI Bio-Layer Interferometry
until further standardization is c
­ onfirmed RT-PCR Reverse transcriptase-­
for each application, it is prudent to test polymerase chain reaction
specific devices before implementing ddPCR Droplet digital polymerase
into procedures. chain reaction
TCIDP50 50% Tissue culture infective
During the preparation of viral vectors dose
for medical studies, a buffer exchange VLP Virus-like particle
after column purification can be
performed with various MWCOs of all
sizes.⁸,⁹,¹⁰,¹⁵ To separate virus particles
from small proteins, a 1,000 kDa cut
off has been shown to work.¹² For the
complete removal of HCV from blood
serum a 30 kDa MWCO has been
­utilized.¹¹ When the assessment of
whole virus content is crucial (e.g. food,
drinking water or marine water samples)
smaller MWCOs (5 – 100 kDa) are
used to ensure full recovery of virus
particles.¹⁹,²¹,²²,²⁵-²⁹

92 Application Notes 4. Concentration and Purification of Viruses

References 16. Guidelines for drinking-water quality - 4th
1. Vannucci, L., Lai, M., Chiuppesi, F., ed. World Health Organization 2011.
­Ceccherini-nelli, L. & Pistello, M. Viral vectors 17. Wyn-Jones, a P. & Sellwood, J. Enteric viruses
: a look back and ahead on gene transfer in the aquatic environment. J. Appl.
­technology. New Microb. 36, 1–22 (2013). ­Microbiol. 91, 945–962 (2001).
2. Luo, D. & Saltzman, W. M. Synthetic DNA 18. Botzenhart, K. Viren im Trinkwasser.
­delivery systems. Nat. Biotechnol. 8, 33–37 Bundesgesundheitsblatt - Gesundheits-
(2000). forsch. - Gesundheitsschutz 50, 296–301
3. Ura, T., Okuda, K. & Shimada, M. Developments (2007).
in Viral Vector-Based Vaccines. Vaccines 2, 19. Lee, K. B., Lee, H., Ha, S. D., Cheon, D. S. &
624–41 (2014). Choi, C. Comparative analysis of viral
4. Mingozzi, F. & High, K. A. Therapeutic in vivo ­concentration methods for detecting the
gene transfer for genetic disease using AAV: HAV genome using real-time RT-PCR am-
progress and challenges. Nat Rev Genet 12, plification. Food Env. Virol. 4, 68–72 (2012).
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Application Notes 4. Concentration and Purification of Viruses 93

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94 Application Notes 4. Concentration and Purification of Viruses

 5. Sartorius Ultrafiltration Products in the Preparation
of Biological Nanoparticles and Medical Nanocarriers
Introduction ­ olecular weight cut-off, MWCO).
m
Paul Ehrlich was inspired by the idea The ­Vivaspin® portfolio spans a volume
of the “magic bullet”* when he for the range from 0.1 to 20 mL, whereas the
first time described in theory toxic Vivaflow® system covers volumes from
drugs assembled to so-called “Nano- 0.1 to 5 liters. Thus, Sartorius offers an
carriers” in 1908.¹ Today, Nanocarriers unrivaled wide range of processable
have found multiple applications in sample volumes, membrane materials
modern medicine and biotechnology. and MWCOs to meet the different
A key application for these special ­requirements of their intended use.
nanomaterials is a targeted delivery Challenges in this context are buffer
of drugs where they act as transport exchange after synthesis, desalting
modules (i. e. as nanoparticles, vesicles, and washing,¹⁰,¹¹ exclusion of solubi-
or micelles) for the active ingredi- lized compounds,¹²,¹³,¹⁴ or aggregates.¹⁵
ent.²,³,⁴,⁵ This is assumed to be more
­effective and less toxic to the (human) Purification is essential to obtain
organism compared to traditionally ­isosmotic conditions for in vivo
administered drug substances.⁶ ­applications, to prevent aggregation
­Besides drug delivery, various further or agglomeration and to remove free
fields using Nanocarriers evolved toxic drugs, ligands, or other sub-
during the last decades; e. g. magnetic strates potentially triggering side
resonance imaging or stem cell gene ­effects. Concentration steps are
therapy with metal-based nano­ ­essential to adjust the amount of
particles,⁷,⁸ or optical imaging with ­pharmaceutical active ingredient in
quantum dots.⁹ the drug and achieve the anticipated
therapeutic or diagnostic effect.
Nanocarriers can be categorized by
their starting material (i. e. metal-, During purification, the separation of
lipid-, polymer-, and protein-based) free substances (starting material)
and by their formation after prepara- from the desired Nanocarriers via size-
tion (i. e. vesicles, particles and mi- exclusion chromatography (SEC) leads
celles). In general, the preparation of to an unavoidable dilution and to the
a nanoparticle suspension or a vesicle necessity of a subsequent concentra-
dispersion in an aqueous medium tion step. In contrast, dialysis purifies
consists of three steps: a) assembly without significant dilution but a con-
of the Nanocarriers (for example, by centration step can still be mandatory,
injections, film hydration, or reverse if higher Nanocarrier concentrations
phase evaporation), b) purification are necessary. Both separation meth-
(for example, by chromatography, ods require quite extensive, costly and
­dialysis or ultrafiltration), and c) timeconsuming manual handling.
­concentration (for example, by This drawback is overcome with the
­ultrafiltration or ­evaporation). ­ultrafiltration utilized by centrifugation
in ­Vivaspin® or with a peristaltic pump
This short review provides examples for the Vivaflow® system. This tech-
of recent literature dealing with the nique is less expensive and quickly
preparation of Nanocarriers. Particular performed with very little manual
focus is laid on the concentration and input. Noteworthy is that purification
purification steps which were per- and concentration steps are performed
formed via ultrafiltration with Sartorius simultaneously.¹⁶
Vivaspin® or Vivaflow® devices with
­different pore sizes (respectively
* In German “Zauberkugel”, opera “Freischütz”
by Carl Maria von Weber

Application Note 5. Ultrafiltration of Nanoparticles 95

 After the Nanocarrier is purified, Prior to performing these different
the determination of drug loading characterizations, a successful
­(conjugation or encapsulation ­purification and concentration of the
­efficiency) is commonly performed. suspension or dispersion is essential.
The conjugation or encapsulation
­efficiency is one of the reference In the following tables you can find
­values to describe and characterize an overview of publications using
Nanocarriers. Other important proper- ­ultrafiltration steps for the purification
ties are the zeta potential and the size and concentration of different kinds
distribution determined via photon of Nanocarriers. Table 2 provides
correlation spectroscopy (PCS), ­guidance on which devices and
high-resolution transmission electron MWCOs to use.
microscopy (HRTEM) imaging, or
­dynamic light scattering (DLS).

Table 1 summarizes examples of Nanocarrier ultrafiltration applications with Sartorius Vivaspin® or Vivaflow®:

Nanocarrier: Size distribution obtained via (HR)TEM or DLS, Application Ref.

Nanoparticle, Vesicle, Micelle Z-Average via PCS and others-if reported

Nanoparticles from metal, metal oxides and functionalized metals

Iron oxides nanoparticles with SD: 4.5 ± 0.9 nm via X-Ray-Diffraction Analysis Magnetic resonance imaging 7
cisplatinbearing polymer coating

Functionalized iron oxide nanoparticles SD: 38 and 40 nm via DLS Stem cell gene therapy and tracking 8

Gold nanoparticles SD: 0.8 – 10.4 nm via Atomic Force Microscopy Antimicrobial activity 17

Protein coated gold nanoparticles SD: 15 and 80 nm via TEM Drug delivery 18

Functionalized gold nanoparticles Core-SD: 2 nm via TEM Targeted imaging tool and antigen delivery 19

Functionalized gadolinium-based Z-Average: 1.1 ± 0.6 nm and 4 – 14 nm Diagnostic and therapeutic application 20, 21
nanoparticles

Functionalized nanocrystals 10 to 20 nm Quantum dots for imaging 9

Nanoparticles from polymers, functionalized polymers and polymersomes

Polymer based Nanoparticles Drug delivery 22

Curdlan coated polymer nanoparticles Z-Average: 280 – 480 nm depending on the Macrophage stimulant activity 23
composition and drug delivery

Docetaxel-carboxymethylcellulose Z-Average: 118 ± 1.8 nm Anti-cancer efficacy studies 4

Polymer Nanoparticles

Functionalized Polymersomes Z-Average: 185 nm Surface functionalization studies 3

96 Application Note 5. Ultrafiltration of Nanoparticles

Lipid Nanoparticles and Liposomes

Liposomes and micelles Z-Average: 100 nm for Liposomes and 15 nm Ischemia-reperfusion injury 25
for micelles

Solid lipid Nanoparticles Z-Average: 100 – 120 nm depending on the Drug delivery (Brain Targeting) 26
used lipid

Bacterial outer membrane vesicles SD: 124 nm via TRPS Tunable resistive pulse sensing (TRPS) 27
Analysis

Bacterial outer membrane vesicles Basic research 28

Bacterial outer membrane vesicles SD: 95 nm Basic research 29

Bacterial outer membrane vesicles SD: 50 – 150 nm via TEM Basic research 30

Liposomes Drug delivery 2

Liposomes Encapsulated hydrophilic drugs (Drug delivery) 31

Micelles

Micelles Drug delivery 4

Hydrophobic drug micelles SD via DLS: 39 – 165 nm depending on Drug delivery 14

based on polymers compound in use

Protein Nanoparticles

Protein Nanoparticles SD: 20 – 40 nm via DLS Drug carrier studies 32

SD = Size distribution

Application Note 5. Ultrafiltration of Nanoparticles 97

Table 2 lists example Sartorius devices and typical MWCOs used for each nanocarrier ultrafiltration application:

Nanocarrier: Sartorius MWCO Ultrafiltration purpose Ref.

Nanoparticle, Vesicle, Micelle Ultrafiltration Device

Nanoparticles from metal, metal oxides and functionalized metals

Iron oxides nanoparticles with Vivaspin® 20 100 kDa Purification and concentration 7
cisplatinbearing polymer coating

Functionalized iron oxide nanoparticles Vivaspin® 20 100 kDa Washing step 8

Gold nanoparticles Vivaspin 20

®
5 kDa Purification step 17

Protein coated gold nanoparticles Vivaspin® 6 10 kDa Separation of Nanoparticles | Dyes and washing 18

Functionalized gold nanoparticles Vivaspin® 10 kDa Purification step 19

Functionalized gadolinium-based Vivaspin® 5 kDa Purification and concentration 20, 21

nanoparticles and 10 kDa

Functionalized nanocrystals Vivaspin® 300 kDa Separation of quantum dots-antibody conjugates 9

and 50 kDa from starting material (prior to enumeration)

Nanoparticles from polymers, functionalized polymers and polymersomes

Polymer based Nanoparticles Vivaspin® 30 kDa Purification and concentration 22

Curdlan coated polymer nanoparticles Vivaspin 20®

3 kDa Washing 23

Docetaxel-carboxymethylcellulose Vivaspin ®
10 kDa Concentration 4
Polymer Nanoparticles

Functionalized Polymersomes Vivaspin® 20 10 kDa Concentration 3

Lipid Nanoparticles and Liposomes

Liposomes and micelles Vivaspin® 20 100 kDa Concentration 25

Solid lipid Nanoparticles Vivaflow® 50 100 kDa Purification 26

Bacterial outer membrane vesicles Vivaflow® 200 100 kDa Buffer exchange and concentration 27

Bacterial outer membrane vesicles Vivaspin® 500 and 20 100 kDa Buffer exchange and concentration 28

Bacterial outer membrane vesicles Vivaflow 200

®
100 kDa Buffer exchange and concentration 29

Bacterial outer membrane vesicles Vivaspin ®

100 kDa Buffer exchange and concentration 30

Liposomes Vivaspin ®
100 kDa External buffer exchange 2

Liposomes Vivaflow 50®

100 kDa Elimination of the free drug 31

Micelles

Micelles Vivaspin® 30 kDa Separation of free substrate and concentration 4

Hydrophobic drug micelles Vivaflow ®

Surfactant removal 14
based on polymers

Protein Nanoparticles

Protein Nanoparticles Vivaspin® 500 3 kDa Separation of the free from the encapsulated drug 32
(Drug binding quantification by subsequent
UV-vis analysis)

98 Application Note 5. Ultrafiltration of Nanoparticles

References 14 Zhang, Y. et al.: Therapeutic surfactant-­
1. Strebhardt, K. & Ullrich, A.: Paul Ehrlich’s stripped frozen micelles. Nat Commun 7,
magic bullet concept: 100 years of progress. 11649 (2016).
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2 Jakoby, J., Beuschlein, F., Mentz, S., Hantel, ­enhancement in THP-1 cells with Gd2O3
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4 Mulder, W. J. M. et al.: Molecular imaging of ­Biofilm Encapsulated Gram-Positive
macrophages in atherosclerotic plaques ­Staphylococci. Small 11, 3183–3193 (2015).
using bimodal PEG-micelles. Magn. Reson. 18 Schäffler, M. et al.: Blood protein coating of
Med. 58, 1164–1170 (2007). gold nanoparticles as potential tool for organ
5 Murthy, S. K.: Nanoparticles in modern medi- targeting. Biomaterials 35, 3455–3466
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Int. J. Nanomedicine 2, 129–41 (2007). 19 Arosio, D. et al.: Effective targeting of
6 Voigt, R. & Fahr, A.: Pharmazeutische ­DC-sign by α-fucosylamide functionalized
­Technologie für Studium und Beruf. gold nanoparticles. Bioconjug. Chem. 25,
Deutscher Apotheker Verlag, 10th Edition ­2244–2251 (2014).
(2010). 20 Miladi, I. et al.: Biodistribution of ultra small
7 Unterweger, H. et al.: Development and gadolinium-based nanoparticles as
­characterization of magnetic iron oxide ­theranostic agent: application to brain
nanoparticles with a cisplatin-bearing ­tumors. J. Biomater. Appl. 28, 385–94
­polymer coating for targeted drug delivery. (2013).
Int J Nanomedicine 9, 3659–3676 (2014). 21 Faure, A. C. et al.: Control of the in vivo
8 Park, W. et al.: Multi-modal transfection ­biodistribution of hybrid nanoparticles with
agent based on monodisperse magnetic different poly(ethylene glycol) coatings.
nanoparticles for stem cell gene delivery Small 5, 2565–2575 (2009).
and tracking. Biomaterials 35, 7239–7247 22 Benita, S., Debotton, N. & Goldstein, D.:
(2014). Nanoparticles for Targeted Delivery of
9 Chalmers, N. I. et al.: Use of quantum dot ­Active Agent. (2008).
­luminescent probes to achieve single-cell 23 Tukulula, M. et al.: Curdlan-conjugated
resolution of human oral bacteria in biofilms. PLGA nanoparticles possess macrophage
Appl. Environ. Microbiol. 73, 630–636 (2007). stimulant activity and drug delivery
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Clarke, S. R. & Voelcker, N. H.: New insights (2015).
into the structure of PAMAM dendrimer | 24 Ernsting, M. J., Tang, W. L., MacCallum, N.
gold nanoparticle nanocomposites. W. & Li, S. D.: Preclinical pharmacokinetic,
­Langmuir 27, 6759–6767 (2011). biodistribution, and anti-cancer efficacy
11 Rademacher, T. & Williams, P.: Nanoparticle-­ studies of a docetaxel-carboxymethyl­
peptide compositions. (2014). cellulose nanoparticle in mouse models.
12 Allard, E. & Larpent, C.: Core-shell type Biomaterials 33, 1445–1454 (2012).
­dually fluorescent polymer nanoparticles for 25 Geelen, T., Paulis, L. E., Coolen, B. F.,
ratiometric pH-sensing. J. Polym. Sci. Part A ­Nicolay, K. & Strijkers, G. J.: Passive target-
Polym. Chem. 46, 6206–6213 (2008). ing of lipid-based nanoparticles to mouse
13 Prach, M., Stone, V. & Proudfoot, L.: Zinc cardiac ischemia-reperfusion injury.
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(2013).

Application Note 5. Ultrafiltration of Nanoparticles 99

 26 Neves, A. R., Queiroz, J. F. & Reis, S.:
Brain-targeted delivery of resveratrol using
solid lipid nanoparticles functionalized with
apolipoprotein E. J. Nanobiotechnology 14,
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derived outer membrane vesicles using
­tunable resistive pulse sensing. Prog.
Biomed. Opt. Imaging – Proc. SPIE 9338,
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28 Blenkiron, C. et al.: Uropathogenic
­Escherichia coli releases extracellular
­vesicles that are associated with RNA.
PLoS One 11, 1–16 (2016).
29 Twu, O. et al.: Trichomonas vaginalis
­Exosomes Deliver Cargo to Host Cells and
Mediate Host:Parasite Interactions. PLoS
Pathog. 9, 22–24 (2013).
30 Tong, T. T., Mörgelin, M., Forsgren, A. &
­Riesbeck, K.: Haemophilus influenzae
­Survival during Complement-Mediated
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31 Prado, J. M. D., Antoranz, J. R. C., Barroeta,
M. Á. E., Barroeta, B. E. & Diaz, M. C.:
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100 Application Note 5. Ultrafiltration of Nanoparticles

 6. Vivaflow® and Vivaspin® Workflow in Protein
­Research Laboratories
Introduction Methods
Efficiency and efficacy of a multiple
cycle experimental procedure was Part 1 – Creating and concentrating
performed using Vivaflow® tangential the culture medium
flow cassettes for initial concentration 2 bottles (4 g) of RPMI-1640 were
and diafiltration of a cell culture super- ­dissolved into 1.8 L dd-H2O and 4 g
natant. This was followed by Vivapure® of sodium acetate was added.
Ion Exchange spin columns for the
­protein purification step and finally The pH was adjusted to 7.2 using 1M
­Vivaspin® 20 ultrafiltration devices for HCl. 2 g of BSA and 1 g of Lysozyme
the final sample concentration and was added as protein samples, meant
­desalting. An artificial mixture of to be separated by chromatography.
­proteins in a RPMI-1640 culture The volume of the cell culture super-
­medium was created to mimic the natant sample was brought up to 2 L
type of product that many researchers using dd-H₂O. After every preparation,
­culture using e.g. the UniVessel device. concentration and purification step, 1
This procedure further reflects a mL sample was set aside for SDS gel
*S
 ample colour
representative only
­method that can be adapted to a analysis.
large number of protein purification
­protocols, selecting alternative Ion exchange chromatography was
MWCOs and device sizes where the method of choice for purifying
­necessary. ­lysozyme from the cell culture
­supernatant, especially from the
­“contaminant” BSA. For this, the 2 L
cell culture supernatant needed to be
concentrated and then diafiltered to
adjust the sample to the starting
­conditions needed for the ion exchange
chromatography binding step.

For concentration and diafiltration, the

Vivaflow® 200 was used with a 5 kDa
MWCO PES membrane. Vivaflow®
200 is a ready-to-use laboratory cross-
flow cassette in an acrylic housing,
which allows caustic cleaning and 4 – 5
re-uses. Two cassettes can be run in
parallel for the concentration of up to
5 L sample volumes. For the 2 L ­sample
to be concentrated in this ­experiment,
one cassette was sufficient. A M
­ asterflex
pump with an ­Easy-Load, size 16 pump
head was used to run the ­Vivaflow® 200
­cassette. Figure 1a. and 1b. show the
­Vivaflow® 200 set up with one or two
cassettes.

Application Note 6. Vivaflow® and Vivaspin® Workflow 101

 The Vivaflow® 200 system was operated Filtrate Volume (mL) Time (hr:min:sec)
at 3 bar. Once 1.8 L of filtrate had been 0 0:00:00
collected, the pump was stopped, the
100 0:03:16
tubes removed from the cell culture
medium concentrate and filtrate and 200 0:06:50

the Vivaflow® system was purged with 300 0:10:45

dd-H₂O. This solution now contained a 400 0:14:38
10-fold concentration of the constitu-
500 0:18:36
ent proteins from the ­original culture
600 0:22:43
medium.
700 0:26:57
A BCA protein detection assay con- 800 0:31:14
veyed a 100% recovery of protein after 900 0:36:01
this first concentration step. Table 1
1,000 0:40:50
­indicates the time needed for the
­sample concentration. 1,100 0:45:46

1,200 0:50:36

1,300 0:55:32

1,400 1:00:24

1,500 1:05:26

1,600 1:10:28

1,700 1:15:52

1,800 1:21:50

Table 1: Vivaflow® 200, 5 kDa MWCO PES

­concentration speed
Figure 1a. Vivaflow® 200 – single cassette

Figure 1b. Vivaflow® 50R – two cassettes

102 Application Note 6. Vivaflow® and Vivaspin® Workflow

 Part 2 – Buffer exchange of culture
medium concentrate
The Vivaflow® 200 system was used for
fast and easy diafiltration. To this end,
the diafiltration reservoir, a Vivaflow®
accessory, was filled with the 200 mL
concentrated sample. Figure 2 shows
the diafiltration set up. The Vivaflow®
200 system was set up as before,
­however attaching an additional tube Figure 2: Diafiltration system set up for buffer
to the diafiltration lid and placing this ­exchange. Culture medium concentrate can
be seen in the center of the image. 1 L 25 mM
new inlet tube into a 25 mM sodium
sodium acetate (exchange buffer) can be seen
acetate (pH 5.5) buffer (needed to connected to the system on the left of the
re-adjust the sample concentrate to image.
the ionic starting conditions of the ion
exchange chromatography step which
was to follow). This enables concentra- Filtrate Volume (mL) Time (hr:min:sec)
tion of the sample in the reservoir to 0 0:00:00
the extent that the original buffer is
100 0:06:58
­removed and collected as waste
­(filtrate). Simultaneously, new buffer 200 0:14:16
(25 mM sodium acetate) is drawn into 300 0:22:39
the closed system, gradually leading to 400 0:29:40
a buffer exchange while keeping the
500 0:37:02
sample volume constant at 200 mL.
600 0:44:15
The system was run at 3 bar. Once 1 L
of buffer had been exchanged, 700 0:51:34
­diafiltration was stopped. 800 0:58:54
900 1:06:03
The 200 mL solution now contained
1,000 1:13:02
the correct buffer to maintain the
­stability of the proteins of interest for Table 2: Diafiltration of 200 mL concentrated
the next part of the protocol and had cell culture supernatant containing the proteins
lysozyme and BSA against 1,000 mL 25 mM
the correct pH and salt concentration
­sodium acetate.
for the ion exchange binding step.
BCA protein quantification again
showed a 100% protein recovery.

Table 2 shows the time needed for

­diafiltration of 200 mL sample against
1,000 mL exchange buffer, again using
Vivaflow® 200 with a 5 kDa MWCO
PES membrane.

Application Note 6. Vivaflow® and Vivaspin® Workflow 103

 Part 3 – Purification of Lysozyme, Two Vivapure® Maxi H S type devices
the protein of interest (Figure 4) were each equilibrated
The purification of Lysozyme was with 10 mL of 25 mM sodium acetate
­performed using a ­Vivapure® cation (pH 5.5), by filling with 10 mL of this
exchange membrane adsorber device buffer and centrifuging for 5 min. in a
(Vivapure® Maxi H S). The membrane swing bucket centrifuge at 500 g and
adsorber matrix holds the active discarding the flow through. Using the
­ligands and performs like a traditional concentrated and buffer exchanged
cation exchanger. However, mem- sample from Part 2, 10 mL samples
brane adsorbers represent a ­special were pipetted into each of the
form of chromatography ­matrix. ­equilibrated Vivapure® devices and
Unlike traditional resins, they make centrifuged again for 5 min. in a
use of convective transport to bring swing bucket centrifuge at 500 g.
proteins to the ion exchange ­surface; The ­Vivapure® devices were washed
hence, binding, washing and elution with 10 mL of 25 mM sodium acetate,
is ­performed quickly and high binding ­discarding the flow through, followed
capacities are achieved, even at high by an elution step with 5 mL of 1 M
flow rates. This allows their use in fast NaCl in 25 mM sodium acetate. A BCA
and convenient centrifugal spin assay revealed 95% lysozyme recovery.
­columns (Figure 3).

Figure 3: Electron micrograph of chromatogra- Figure 4: Vivapure® Maxi spin columns can be
phy gel beads (upper right) in comparison to a used in a centrifuge for fast and easy protein
Q ion exchange membrane adsorber (back- purification.
ground), revealing 100-fold larger pore sizes of
the membrane adsorber.

104 Application Note 6. Vivaflow® and Vivaspin® Workflow

 The eluate was then concentrated in Part 4 – Analyzing the samples
a Vivaspin® 20 (5 kDa MWCO PES), The samples of the individual steps
­Figure 5, and centrifuged at 5,000 g were analyzed by SDS gel, using
for 10 min. or until approximately 2 mL ­reducing sample buffer (prepared by
of concentrate had been collected. adding 50 μL 2-mercaptoethanol to
The device was then re-filled with 950 μL Laemmli sample buffer). For all
18 mL 50 mM potassium phosphate steps, 5 μL of the 1 mL sample taken
buffer (pH 7.2) to 20 mL for a final during the experiment were diluted
­buffer exchange and desalting of the with 95 μL reducing sample buffer, of
purified sample. The sample was again which 20 μL were loaded onto a 12%
centrifuged until a final sample volume Tris-HCl SDS gel (Figure 6).
of 2 mL had been attained. A BCA
assay revealed 97% lysozyme recovery.

Figure 6: Coomassie stained 12% Tris-HCl SDS

gel loaded with 20 μL sample preparations.
Lane 1: Marker (SDS Broad Range); Lane 2:
Original sample; Lane 3: Original sample filtrate
(Part 1); Lane 4: Marker; Lane 5: Buffer exchange
concentrate (Part 2); Lane 6: Filtrate after
Figure 5: Vivaspin® 20 ultrafiltration device, on ­binding (Part 3); Lane 7: Marker; Lane 8: Filtrate
the right with a pressure cap which allows pres- after elution (Part 3); Lane 9: Filtrate after
surization of the device as an alternative to the ­concentrating and desalting (Part 3); Lane 10:
regular centrifugal operation. Concentrate after concentrating and desalting.

Application Note 6. Vivaflow® and Vivaspin® Workflow 105

 Conclusion Task Time Recovery
The overall result shows that a stan- Vivaflow® 200 1 hour 25 min. 100%
dard and straightforward procedure set up and
­concentration
can be followed to concentrate, purify,
isolate and analyze a protein of interest Vivaflow® 200 1 hour 20 min. 100%
set up and
from a cell culture, using Vivaflow®
­diafiltration
200 tangential flow units for cell
­culture supernatant concentration Vivapure® 45 min. 95%
­purification
and diafiltration, Vivapure® for ion
­exchange chromatography, followed Vivaspin® 30 min. 97%
­concentration
by Vivaspin® 20 for final sample and desalting
­concentration and desalting.
Total 3 hours 45 min. 92%

In many cases dialysis, which is an

overnight procedure would be per- Products used in this Order No.
formed instead of the much quicker ­experiment
alternative - ultrafiltration. Here, we Vivaflow® 200, VF20P1
show how time-saving and efficient 5 kDa MWCO PES
­ultrafiltration is for diafiltration and 500 mL Diafiltration Reservoir VFA006
­desalting applications, as well as for Vivapure® S H Maxi VS-IX20SH08
protein concentration.
Vivaspin 20,
®
VS2011
5 kDa MWCO PES
The set up and completion of protein
purification takes approx. 3.45 h using Table 3: Processing times for a complete
this method, starting from a culture ­protein purification workflow.
supernatant, with high protein recov-
eries in each step (see Table 3). A total
protein purification procedure can
therefore be completed within 1 ­working
day, including SDS gel analysis, ­utilizing
this strategy, when adapted to
­individual needs.

106 Application Note 6. Vivaflow® and Vivaspin® Workflow

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Status: 05| 2022