CLEANING, DECONTAMINATION AND
SANITATION
Dr. Shatavari Kulshrestha
Cleaning: the removal of ‘dirt’, i.e. miscellaneous organic and
inorganic material which may accumulate in process areas or
equipment during production
Decontamination: the inactivation and removal of undesirable
substances, which generally exhibit some specific biological
activity likely to be detrimental to the health of patients receiving
the drug
e.g. endotoxins, viruses, or prions
Sanitization: the destruction and removal of viable microorganisms (i.e.
bioburden)
CDS procedure is routinely applied to
1) surfaces in the immediate manufacturing area which do not come
into direct contact with the product (e.g. clean room walls and floors,
work tops, ancillary equipment)
2) surfaces coming into direct contact with the product (e.g.
manufacturing vessels, chromatographic columns, product filters, etc.)
CDS OF THE GENERAL MANUFACTURING AREA
Primary cleaning
scrubbing/rinsing the target surface with water or a detergent
solution
Dirt can inactivate many disinfectants or shield microorganisms
from disinfectant action
Decontamination/sanitation procedures
disinfectants or other bacteriocidal agents
Commercially available disinfectants containing active ingredients
including alcohols, phenol, chlorine and iodine
Use of different disinfectant on rotating basis, to minimize the likelihood of
the development of disinfectant-resistant microbial strains
• CDS of clean room walls, floors and accessible surfaces of clean
room equipment is routinely undertaken between production
runs
Fogging the room
This is achieved by placing some of the disinfectant in an
aerosol-generating device (a ‘fogging machine’)
This generates a fine disinfectant mist, or fog, within the clean
room, capable of penetrating areas difficult to reach in any
other manner
CDS OF PROCESS EQUIPMENT
• CDS of surfaces/equipment coming into direct contact with the
product requires special consideration
no trace of the CDS agents subsequently remain on such surfaces,
as this would result in automatic product contamination
The final stage of most CDS procedures to such process
equipment, involves exhaustive rinsing with highly pure water
(water for injections; WFI, followed if at all possible by autoclaving
• CDS of processing and holding vessels, as well as equipment that
is easily detachable/ dismantled (e.g. homogenizers, centrifuge
rotors, flexible tubing filter housing, etc.): Easy
• CDS of large equipment/process fixtures can be more
challenging, due to the impracticality/undesirability of their
dismantling
• Examples include the internal surfaces of fermentation
equipment, large processing/storage tanks, process-scale
chromatographic columns, fixed piping through which product is
pumped, etc.
• Specific ‘cleaning in place’ (CIP) procedures
A detergent solution can be pumped through fixed
pipework, followed by WFI and then the passage of
sterilizing ‘live’ steam generated from WFI
Internal surfaces of fermentation/processing vessels :
scrubbed down
Passage of hot or cold water as appropriate through
jackets
Passage of steam through the jacket of the empty
vessel facilitates sterilization of its internal surfaces by
dry heat
The cleaning of process-scale chromatography (purification of
biopharmaceuticals)
CIP protocols must thus be applied periodically to such systems
The level and frequency of CIP undertaken will depend largely on the level
and type of contaminants present in the product-stream applied
Columns used during the earlier stages of purification may require more
frequent attention than systems used as a final ‘clean-up’ step of a nearly
pure protein product
While each column is flushed with buffer after each production run, a full-
scale CIP procedure may be required only after every 3–10 column runs
Contamination in chromatographic column
Product derived from microbial sources :
contamination of chromatographic media with lipid, endotoxins, nucleic acids and
other biomolecules
Plant-derived extracts
column fouling with pigments and negatively charged polyphenolics, various substances
released from plant cell vacuoles (many of which are powerful protein
precipitants/denaturants)
some plant-derived enzymes are capable of degrading certain carbohydrate-based
(e.g. dextran) chromatographic media
Extracts from animal/human tissue
column contamination with infectious agents or biomolecules, such as lipids
Buffer components may sometimes precipitate out of solution within the column
Application of concentrated solutions of neutral salts (e.g. KCl or NaCl)
• effective in removing precipitated/aggregated proteins, or other material
retained in the column via ionic interaction with the media
Buffers containing EDTA or other chelating agents
• remove any metal ions associated with the gel
Detergent solutions (dilute)
• removing lipid and a whole range of other contaminants, Solvent-containing
(e.g. ethanol, butanol, isopropanol) buffers may also be used in this regard
Increasing the column temperature to 50–60℃
may sometimes be considered, particularly if lipid appears to be a major
column contaminant (most lipids liquefy at such temperatures)
Sodium hydroxide
• one of the most extensively used chromatography CIP agents
• It is readily available, inexpensive and effective. It is usually applied to
a column at strengths of up to 1.0 M
• At such concentrations, it quickly removes/destroys most
contaminants, including microorganisms and viruses. It will also
degrade endotoxin within minutes
• Most types of chromatographic media can withstand incubation with NaOH
for prolonged periods. This allows CDS efficiency to be maximized by
retaining NaOH in the column for time periods of the order of 30–60 minutes
(silica gel is an exception, as it is quickly destroyed at pH values greater than
8)
• The chromatographic column is subsequently rinsed exhaustively by
pumping WFI (or buffer made with WFI) through, until the column effluent is
free from all traces of NaOH
• Prolonged exposure of the chromatographic media/column parts to residual
NaOH could promote column deterioration, and obviously could
contaminate/inactivate the protein stream in the next production run
• Internal surfaces of the column, its valves and piping, are smooth, impervious
and devoid of recesses which could harbour microorganisms or other
contaminants
• Periodic system disassembly allows more extensive CDS procedures to be
undertaken. Most columns are manufactured from glass, or more usually, tough
plastic or stainless steel
• After a thorough cleaning of all disassembled components, sterilization by
autoclaving is usually undertaken prior to re-assembly. Most chromatographic
media likewise can be autoclaved before column re-pouring
CIP of the ring main systems used to store and circulate WFI and purified
water around the pharmaceutical plant is also routinely undertaken
This normally entails emptying the ring main systems (including
reservoirs), opening all the outlet valves, and subsequently pumping
sterile steam through all pipework
This is generally sufficient to physically dislodge any traces of trapped
particulate matter or biological agents harboured in the system
