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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.), is usually relatively straightforward. However, 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 can generally be used to accommodate such equipment. 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 can be scrubbed down. Such vessels are generally jacketed (Figure 3.4), thus allowing temperature control of their contents by passage of cooling water/steam through the jacket, as appropriate. 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 systems used in the purification of biopharmaceuticals can also present challenges. Although such systems are disassembled periodically, this is not routinely undertaken after each production run. 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. Most of the contaminants present in such columns are acquired from these previous production runs.
THE DRUG MANUFACTURING PROCESS 103
Figure 3.4. Diagram of a typical jacketed processing vessel. Such vessels are usually made from high grade stainless steel. By opening/closing the appropriate valves, steam or cold water can be circulated through the jacket. In this way, the vessel’s contents can be heated or cooled, as appropriate. In addition, passage of steam through the jacket of the empty vessel will effectively sanitize its internal surfaces
Processing of product derived from microbial sources can result in contamination of chromatographic media with lipid, endotoxins, nucleic acids and other biomolecules. Application of plant-derived extracts can result in column fouling with pigments and negatively charged polyphenolics, as well as various substances released from plant cell vacuoles (many of which are powerful protein precipitants/denaturants). In addition, some plant-derived enzymes are capable of degrading certain carbohydrate-based (e.g. dextran) chromatographic media. Chromatography of extracts from animal/human tissue can result in column contamination with infectious agents or biomolecules, such as lipids. Furthermore, buffer components may sometimes precipitate out of solution within the column.
Fortunately, most types of modern chromatographic media are resistant to a range of harsh physicochemical influences that may be employed in CIP protocols (Table 3.6). CIP protocols for chromatography columns are normally multistep, consisting of sequential flushing of the gel with a series of CDS agents.
Table 3.6. The range of CIP agents often used to clean/sanitize chromatographic columns. Most CIP protocols would make use of two or more of these agents, allowing them to sequentially percolate through the column at a slow flow rate. Contact time can range from several minutes to overnight. NaOH is particularly effective at removing most contaminant types
Non-ionic detergents (0.1-1.0%) NaOH (0.1-1.0M)
Acetic acid (20-50%)
EDTA (—1.0 mM)
Protease solution Dilute buffer
Application of concentrated solutions of neutral salts (e.g. KCl or NaCl) is often effective in removing precipitated/aggregated proteins, or other material retained in the column via ionic interaction with the media. The use of buffers containing EDTA or other chelating agents helps remove any metal ions associated with the gel. Use of (dilute) detergent solutions is effective in 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°C may sometimes be considered, particularly if lipid appears to be a major column contaminant (most lipids liquefy at such temperatures).
Sodium hydroxide is 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 (discussed later) within minutes.