Manufacturing processes for biotherapeutics and vaccines require efficient cleaning, sanitization, and sterilization procedures to guarantee product safety. Such procedures are an integrated part of all steps of manufacturing and should be validated for their efficacy.
Cleaning is the procedure where impurities (both product-related [fragments, aggregates, etc.] and process-related [HCP, etc.]) and contaminants (from raw material, microbes, etc.) are removed to minimize carryover to the next cycle of a process. Cleaning is also a means to increase the life length of filters and resins. Cleaning is most often performed without disassembling the equipment and is thus referred to as Cleaning-in-Place (CIP). Sanitization is used to reduce bioburden (viruses, bacteria, and spores) to a pre-determined level. Sanitization does not necessarily lead to the absence of living organisms. Sterilization is a procedure that completely eliminates living microorganisms including spores.
In manufacturing processes, both cleaning and sanitization must be addressed. To minimize problems not related directly to the feedstream, the use of pyrogen-free, high quality water (e.g., water for injection, WFI), filtered air, and highquality grade solvents and buffers, is important.
Cleaning and sanitizing reagents, their method of preparation, concentration, volume, contact time and temperature vary significantly depending on whether a bioreactor, filtration membrane, or packed chromatography column is being considered. The selection of cleaning and sanitizing agents will be dictated by effectiveness, chemical compatibility, costs, waste disposal, and experience.
Regulatory bodies like the FDA and EMA are stressing the importance of efficient cleaning and sanitization methods. Validation of these methods’ efficacy is equally important. Furthermore, standard operating procedures (SOP) including acceptable ranges are needed.
BASIC CONSIDERATIONS
Cleaning is necessary to avoid carryover between cycles and batches in a manufacturing process. A cleaning step should be performed after each cycle to avoid accumulation of impurities/contaminants which may later be released into the product stream. CIP will allow the extended reuse of consumables like filtration membranes and chromatography resins. Incomplete cleaning will affect the functionality of resins and filters and reduce their lifespan. The economic impact will be significant if the lifetime of the consumable in production can be increased.
A bioburden incident will lead to a stop in production followed by a quality assurance (QA) investigation to find the root cause of the bacterial contamination. The biopharmaceutical production lot and/or the chromatography resin may need to be discarded. This will be very costly and have a major commercial impact. Efficient sanitization procedures will significantly reduce the risk for transferring living organisms between cycles and batches. Reduction of microbial contamination will also minimize the risk of product degradation by proteolysis. Sanitization is often combined with the cleaning procedure, provided the equipment and consumables are compatible with the chemical agents. Sterilization is almost exclusively used for bioreactors to avoid microbial infection. Certain filter cassettes and resins can be sterilized prior to long term storage
Impurities and Contaminants in the Bioprocess
During a biopharmaceutical manufacturing process, the equipment and consumables will be exposed to a large number of impurities and contaminants that need to be removed by CIP and/or sanitization.
In the purification process, impurities can be divided into two groups: product-related impurities and process-related impurities. Product-related impurities include product fragments, modified product, aggregates, etc. Process-related impurities are host cell proteins (HCP), DNA, lipids, endotoxins, etc. Contaminants are considered to originate from the outside and are unintentionally introduced into the process. Contaminants can be introduced from raw materials and may include microbes, viruses, and chemical additives like antifoams. From a next-batch preparation perspective (and for equipment and column integrity), it is relevant to consider all remains of impurities and contaminants, including residual target protein/ biomolecule, as contaminants.
The nature of the feedstream (e.g., whether the target molecule is produced by secreting mammalian cells or purified from E. coli homogenate) will lead to different cleaning challenges in the downstream process. In the former example, once the cells have been harvested the target molecule is relatively pure compared to the latter, which consists of significantly higher levels of cell debris, HCP, DNA, and endotoxins. Modifications of feedstreams (e.g., additives for optimization of upstream production) can also cause downstream cleaning challenges.
From a CIP/sanitization point, transmissible spongiform encephalopathy (TSE) agents can be considered a special case. The TSE agent is very stable and maintains its infectivity even under harsh sanitization conditions, although certain chemical treatments will reduce or eliminate TSE agents. Proper sourcing of ruminant raw materials will decrease the risk of introduction of TSE agents.
The great variety of contaminants from the process may require a multitude of different CIP/sanitization methods. For example, microorganisms and DNA are more likely to be the major contaminants in the early steps of a manufacturing process, while contamination from product-related impurities, including the target protein, are common in later stages.
Methods for CIP/sanitization need to be included early in the process development work. During development of cleaning procedures, it is important to use a feed material with cell and product concentrations relevant to the final process to obtain reliable results. Initial cleaning experiments for chromatography resins can be performed under static conditions in microwell plates first to gain an understanding of critical issues and the cleaning effectiveness of chemicals. Evaluation of cleaning conditions can also be done in parallel using miniaturized microliter scale chromatography columns operated by a liquid handling system. Verification and further development of a cleaning procedure is done at lab scale and during subsequent process scale up. Validation of its efficiency by chemical, biochemical, and biological assays should be done in parallel. It is important to investigate early in development how the cleaning process will affect the lifespan of filters and resins.
Microorganisms originate from water, soil, air, plants, animals, and humans and can enter the process through various routes. Operators working in the manufacturing areas are a major potential source of contamination. Other potential sources include facility, equipment, process operations, raw materials, column resins, filter membranes, process gases, and water. Bioburden (i.e., bacteria and fungi) measurements are problematic. There may be assay interference from the environment in which the sample is taken; therefore, bacteriostasis and fungistasis testing are necessary. Both the numbers and types of microorganisms found are relevant. Speciation is important as a change in the type of organism can present an untested challenge that requires process validation. Even if there is a very low level, which may be acceptable, there is concern that the organism might grow during processing. Furthermore, regulatory authorities’ requirements on bioburden control are steadily increasing.
A successful sanitization process where no living organisms can be detected does not indicate that the cleaning requirements have been achieved. There might still be dead microorganisms and impurities like HCP, DNA, exotoxins, and endotoxins remaining.
Cleaning Agents
Common agents for removal of impurities are shown in Table 33.2. Sodium hydroxide (NaOH) is by far the most used CIP chemical. It is effective for a variety of impurities, is easy to detect, and has a low cost. It is often used at a concentration of 1M. High concentrations of NaOH can denature remaining contaminants and make them more difficult to remove. It is therefore important to elute, wash out, and strip off bound material in order to regenerate a chromatography resin before introducing, for example, 1 M NaOH. A strip or regeneration step can be low pH for an affinity resin or high salt (1.0 M NaCl) for an IEC resin. Some material used in the manufacturing process (affinity ligands, filters, O-rings, etc.) may not withstand 1M NaOH. A lower concentration of NaOH including 1 M NaCl to maintain a high ionic strength might still be effective. An alternative approach is to perform the cleaning using acetic acid or phosphoric acid at pH 3.
If neither high nor low pH can be used for cleaning, one possibility is to use 6 M guanidinium hydrochloride (Gua-HCl) or 8M urea. Also 8M urea, 1.0 M NaCl at pH 2.5 has proven effective for removal of discoloration from an AIEC (anion exchange chromatography) column.
6M Gua-HCl is corrosive to stainless steel, has a high cost and disposal issues, and is only recommended as a last resort. Mixtures of NaOH and NaCl are often used effectively to remove DNA. Deoxyribonuclease (DNase) is sometimes introduced to remove DNA, but the enzyme material suitable for biopharmaceutical manufacturing is expensive and requires analytical methods to confirm its removal after cleaning. It should be noted that chloride ions are corrosive for stainless steel, especially in combination with low pH, and should therefore be washed out immediately after completion of a CIP cycle. Sodium sulphate is a much less corrosive alternative to NaCl.
Lipid and lipoprotein impurities may in some cases require the use of organic solvents for complete removal. The most frequently used solvents are 20% ethanol and 30% isopropanol. Other effective cleaning agents are acetonitrile and nonionic detergents. Handling and dilution of larger quantities of solvents beyond the laboratory scale may require explosionproof facilities. However, diluted solvents under certain concentrations and volumes can most often be handled in ordinary manufacturing facilities. It is important to know which local regulations apply for such operations. Many biopharmaceutical companies strive to find alternatives to alcohol and other solvents in their production.
Endotoxins are a group of impurities with strong physiological effects, and are therefore important to remove. Endotoxins are produced by gram-negative bacteria and constitute a particular problem when the feed material originates from E. coli fermentation. Inactivation and removal of endotoxins can be achieved by NaOH treatment. The effect is dependent on concentration and contact time. For example, 5h treatment with 1M NaOH at room temperature reduces the endotoxin to an acceptable level.
The efficiency of the CIP method used needs to be carefully tested and validated to ensure that both the pre-determined levels of residual contaminants are reached and all cleaning agents are removed. The Total Organic Carbon (TOC) method detect leaking impurities in rinsing solutions and is the most frequently used analytical method. Modern TOC instruments are available for continuous measurements and on-line evaluation. For more specific measurements of HCP and product-related impurities, highly sensitive immunoassays can be used. Other useful techniques for detection of impurities are SDS-PAGE and HPLC. Detection of remaining cleaning agents like salts, alkali, and acids can be done by conductivity or pH measurements. Determination of residual solvents is most often done by gas chromatography. Sanitization Agents
The effectiveness of a sanitization procedure is investigated by challenging the system with certain representative microorganisms and studying the reduction. A typical challenge study uses the American Type Culture Collection (ATCC) microorganisms that are used to test WFI. Those include, for example, E. coli (gram-negative bacteria), S. aureus (gram-positive bacteria), C. albicans (yeast), and A. niger (mold).
NaOH is the most commonly used sanitizing agent. In cases where the system can tolerate harsher conditions, oxidizing chemicals like sodium hypochlorite, hydrogen peroxide, or peracetic acid are even more effective. Concentration, contact time, and temperature are factors which are always important to investigate.
Detection of remaining colony forming units (CFU) after sanitization is done for rinsing solutions and exposed surfaces. A reduction of 106 CFU is most often acceptable. Detection of certain species of microorganisms by traditional agar plates may take several days before an accurate result is obtained. This means that it will take days before a new processing cycle can start to avoid any remaining risk of contamination. To significantly reduce cycle time, it is possible to use rapid microbiological methods (RMM) as an alternative to agar plates. The use of such methods is supported by regulatory authorities but requires appropriate validation to demonstrate that they are equal to or better than traditional methods. Numerous RMM techniques are supplied from several vendors and have been implemented or reviewed by the industry. Viral contaminations are usually removed by sodium hydroxide (0.1–1M) or acetic acid (0.1–1 M). As viruses constitute a group of organisms with a wide range of stabilities, it is important to determine the demands of the decontamination process case by case.
Spores are much more resistant towards chemical treatment than vegetative microorganisms. A cleaning process using 60% ethanol in 0.5M acetic acid followed by 0.2M NaOH for 3h was shown to reduce B. subtilis below the level of detection. An alternative that is less aggressive against equipment, filters and resins, but still effective in killing spores, is a mixture of 120mM phosphoric acid, 167mM acetic acid, 2.2% benzyl alcohol, referred to as PAB. For equipment and materials that can withstand oxidizing agents, treatment with peracetic acid solutions has been shown to kill bacterial spores effectively.
