Upstream and Downstream Processing
ANIMAL CELL CULTURE
Media formulation / optimization for microbial,
baculoviral and mammalian cell culture projects.
• Non-animal origin media solutions
• Development of fed-batch strategies for
higher cell densities and product titers
•
BIOPHARMACEUTICALS
Clear idea of product
• Selection of producing organism
• Strain screening & Improvement (Molecular)
• Formulation medium requirements
• Medium optimization
OIL & GAS
• Producing Well (Onshore or Offshore)
• searching for and the recovery and production
of crude oil and natural gas
Upstream Process - Known as exploration and production (E&P)
sector.
BIOPROCESS
• Isolation and screening of microbes
• Strain improvement
• Inoculum development
• Medium formulation and optimization
• Fermentation process (Batch, Fed-Batch &
Continuous)
USP DSP
Bioprocessing of Cloned Strain
USP
DSP
Bioprocessing of Recombinant Protein
Definition of Upstream
The upstream stage of the production process involves searching
and extracting raw materials.
The upstream part of the production process does not do anything
with the material itself, such as processing the material.
This part of the process simply finds and extracts the raw material.
Thus, any industry that relies on the extraction of raw materials
commonly has an upstream stage in its production process.
In a more general sense, "upstream" can also refer to any part of the
production process relating to the extraction stages.
Cont.
The upstream part of a bioprocess refers to the FIRST STEP in which
microbes/cells are grown, e.g. bacterial or mammalian cell lines (see Cell culture),
in bioreactors.
Basically upstream processing involve all those steps related with inoculum
development, media development, improvement of inoculum by genetic
engineering process, optimization of growth kinetics so that product
development can improve tremendously.
Fermentation has two part upstream and downstream. after product development
the next step is purification of product for desired quality.
When the system reached the DESIRED DENSITY (for batch and fed batch
cultures), they are harvested and moved to the downstream section of the
bioprocess.
ISOLATION
Isolation » involves obtaining either pure or mixed cultures followed by
assessment to determine which carry out the desired reaction or product.
Possible to design the isolation procedure to encourage the producers » eg:
selective media.
Criteria to choose an organisms;
1. Nutritional characteristics ~ cheap or pre-determined
2. Optimum temperature ~ >40˚C (cooling cost – large scale)
3. Reaction and suitability to the system employed ~ shear stress (high agitation)
4. Stability and amenability to genetic manipulation ~ being modified
5. Ease of product recovery ~ cost effective (reduce downstream processes)
Major Culture Collections
Culture collection Address
American Type Culture Collection (ATCC) United State of America (USA)
Deutsche Sammlung
Zelkulturen (DSM)
von
Mikroorganismen uud Germany
Japan Collection of Microorganisms (JCM) Saitama, Japan
Isolation methods :
1. Enrichment Liquid Culture
- The selective re-established by inoculating the enriched
medium to fresh medium. Microbe
- Several sub-culturing to obtain pure culture. Selective media
2. Enrichment Solidified Culture Clear zone
Commercial
- Involves the use of selective medium incorporating the substrate
substrate of the enzyme. Paper Disc
- Commonly used » clearing zone
SCREENING
Antibiotics – resistance
OR
Provision of test organisms
- Increased sensitivity of resistances
Gene cloning
- Coding for specific enzymes or receptors
Development of reporter gene assays
- used to detect activation of control sequence
Molecular probes
- Enable the detection of organisms to produce
specific products
Development of immunologically based assay
- Such as ELISA (very specific)
PRESERVATION
Slant Agar
1. Storage at reduced temperature
Agar Slopes
- On slant agar ≈ approx. 6 months
Liquid nitrogen
- Reduce metabolic activities (dormant)
- Suspend in cryoprotective agent » 10% glycerol
Liquid Nitrogen
2. Storage in a dehydrated form
Dried cultures
- Dried soil samples ≈ approx. 2 weeks (-5ºC)
Lyophilization
- Freeze drying (microbe + coating reagents ≈ many years)
Freeze-dried
Downstream Processing
Raw
Materials
DSP
Extraction of Plant Materials
UPS
Development
& Evaluation
Bioseparation of Vaccines
Extraction on Transgenic Systems
DSP
USP
Bioprocessing of Lipase
PARAMETER VARIANTS
Process analytics as understanding of the RELATIONSHIP between process,
product(-variants) and contaminants. During upstream and downstream
processing, deviations from the optimal process parameters (IN RED) for each of
the individual steps will lead to the emergence of the product variants T, F, MO,
M, D, and A (in blue, symbols see Figure 1) and contaminants (in green) which are
difficult to detect in the final product by routine quality control. These may affect
the production costs, recovery, efficacy and safety of the protein drug in
differential fashion with safety being of highest importance.
TOTAL PRODUCTION COST
60%
DOWNSTREAM PROCESSING
Downstream processing refers to the recovery and purification of
biosynthetic products, particularly pharmaceuticals, from natural
sources such as animal or plant tissue or fermentation broth,
including the recycling of salvageable components and the proper
treatment and disposal of waste.
It is an ESSENTIAL STEP in the manufacture of pharmaceuticals
such as antibiotics, hormones (e.g. insulin and human growth
hormone), antibodies and vaccines; antibodies and enzymes used
in diagnostics; industrial enzymes; natural fragrance and flavor
compounds.
Downstream processing is usually considered a specialized field in
biochemical engineering, itself a specialization within chemical
engineering, though many of the key technologies were developed
by chemists and biologists for laboratory-scale separation of
biological products.
Downstream processing and analytical bioseparation both refer to
the separation or purification of biological products, but at
different scales of operation and for different purposes.
Downstream processing implies manufacture of a purified product
fit for a specific use, generally in marketable quantities, while
analytical bioseparation refers to purification for the sole purpose
of measuring a component or components of a mixture, and may
deal with sample sizes as small as a single cell.
STAGES IN DOWNSTREAM
RIPP Scheme
1. REMOVAL OF INSOLUBLES
• The first step and involves the capture of the product as a solute in a particulate-free liquid,
for example the separation of cells, cell debris or other particulate matter from fermentation
broth containing an antibiotic.
• Typical operations to achieve this are filtration, centrifugation, sedimentation,
precipitation, flocculation, electro-precipitation, and gravity settling.
2. ISOLATION OF PRODUCTS
• The removal of those components whose properties vary markedly from that of the desired
product.
• Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit
operations involved.
3. PURIFICATION OF PRODUCTS
• Done to separate those contaminants that resemble the product very closely in physical and
chemical properties.
• This stage contributes a significant fraction of the entire downstream processing expenditure.
• Examples of operations include affinity, size exclusion, reversed
phase chromatography, crystallization and fractional precipitation.
4. POLISHING
• Describes the final processing steps which end with packaging of the product in a form that
is stable, easily transportable and convenient.
• Crystallization, desiccation,lyophilization and spray drying are typical unit operations.
Extraction
The process of obtaining something from a mixture of
compounds/cells/tissues by chemical or physical or
mechanical approaches.
• When the extraction takes place from one liquid medium to
another, the process is referred to as liquid-liquid extraction.
• When a liquid is used to extract solutes from solid material, the
process is referred to as solid-liquid extraction.
• Extracting minerals from solids by dissolving them in a liquid is
called leaching.
Biological things to be
extracted
Nucleic acids
Proteins
Enzymes
Lipids
Secondary metabolites
Bioseparation
“… After successful fermentation or enzyme reactions, desired
products must be separated and purified. This final step is
commonly known as downstream processing or bioseparation,
which can account for up to 60% of the total production
costs, excluding the cost of the purchased raw materials…”
What is separated in
Bioseparation?
Biological derived products can be classified based on:
a) chemical nature (Table 1.1)
b) application (Table 1.2)
Nature of
Bioseparation
Biological products are present in very low concentrations
Bioseparation has to be very selective in nature
Stringent quality requirements for products
Biological products are susceptible to denaturation and other
forms of degradation
Many biological products are thermolabile and hence many
bioseparation techniques are usually carried out at sub-
ambient temperatures
Bioseparation is frequently based on multi-technique
separation
Characteristic of
Bioseparation
• Variety of products to be separated: Antibiotics, amino
acids, enzymes, organic acid and solvents, vitamins,
yeasts, growth factors, nucleotides, dextrans…
• Broad spectrum of separation method can be used:
Physical separations, equilibrium controlled separations,
rate controlled separations
• Mode of operations: steady and unsteady states, batch
and continuous, co-current and counter current
• Scale of operation: laboratory, pilot plant, large scale
Basic of Separations
Factor Types of Separation
Size Filtration, Membrane Separation, Centrifugation
Density Centrifugation, Sedimentation, Floatation
Diffusivity Membrane Separation
Shape Centrifugation, Filtration, Sedimentation
Polarity Extraction, Chromatography, Adsorption
Solubility Extraction, Precipitation, Crystallization
Electrostatic Adsorption, Membrane Separation, Electrophoresis
Charge
Volatility Distillation, Membrane Distillation
Fundamental Principles
Mechanical Physical Thermal Chemical
•Filtration •Absorption •Drying Chemical
•Centrifugation •Adsorption •Distillation reaction
•Membranes •Crystallization •Rectification
•Classification •Precipitation •Evaporation
•Purification •Extraction
•Agglomeration
•Washing
Types of Separation
1. Gas-liquid separation
- Absorption
2. Vapor-liquid separation
- Distillation
3. Liquid-liquid separation
– Extraction
4. Fluid–solid separation
– Adsorption, Leaching, Chromatography, Crystallization
5. Mechanical-physical separation
– Precipitation, Settling & Sedimentation, Centrifugation,
Filtration, Membrane Separation.
Bioseparation Techniques
Low-resolution + High-resolution +
high-throughput low-throughput
Cell Disruption Ultracentrifugation
Precipitation Chromatography
Centrifugation Affinity separation
Liquid-liquid Extraction Electrophoresis
Leaching
Filtration
Supercritical Fluid Extraction
Microfiltration
Ultrafiltration
Adsorption
Crystallization
