Large scale production of biologics is susceptible to microbial contamination. Bioburden contamina-tions of non-sterile process intermediates represent a risk to patient safety and product quality. Even after bioburden removal by 0.2 μm filtration, and even if both Drug Substance and Drug Product spec-ifications are met, subcellular microbial components like toxins, lipopeptide/lipoproteins, flagellin, bacterial and fungal DNA, cell wall polysaccharides, extracellular proteases or endoglycosidases re-main in the product. Those microbial components potentially lead to toxic, allergic or inflammatory responses in humans or product degradation or modification. The CCAB approach described here enables a comprehensive assessment of these risks.
Bioburden testing is an important part of pharmaceutical microbiology and provides data in relation to the quality of pharmaceutical products during manufacture. Little guidance is provided in relation to test methodology, culture media and incubation parameters. The quality control laboratory, therefore, needs to establish the most appropriate method. This paper outlines a case study for the selection of incubation parameters for the bioburden assessment of in-process samples using the Total Viable Count technique and pour plate method. While the outcome of the exper ment con-tained within the paper relates to a specific set of processes, the approach taken can be used by oth-er laboratories to compare or to develop their test methods and techniques for bioburden determi-nations.
Microbial control during the drug substance and drug product manufacturing process is critical for ensuring product quality and safety. For sterile biological drug products (finished dosage forms) typi-cally manufactured by sterile filtration followed by aseptic processing, control of the microbial load at the sterile filtration step is an important component of the overall microbial control strategy. Both FDA and EMA regulatory guidelines stipulate that a maximum acceptable bioburden level, which is referred to as a pre-filtration bioburden level in this paper, should be stated at the point immediately prior to the sterile filtration step. The EMA guideline further states that a bioburden limit of no more than 10 colony-forming units (CFU) per 100 mL will be considered acceptable in most situations. The EMA guideline also states that a pre-filtration sample volume of less than 100 mL may be tested if justified. This paper introduces a risk-based method to establish pre-filtration bioburden acceptance levels and alternative test volumes. The relationship between bioburden risk, pre-filtration biobur-den test limits, and sterile filtration process parameters, such as filtration volume, filter surface area, and microbial retention capacity of the sterilizing filter, was statistically determined. Taking into ac-count the batch filtration volume, it is shown that pre-filtration bioburden test volumes and ac-ceptance limits other than 10 CFU/100 mL may be justified, without compromise to sterility assur-ance.
According to the FDA Guidance for Industry (2004), bioburden is defined as follows: “The total num-ber of microorganisms associated with a specific item prior to sterilization.
”Another definition of bioburden is given by EudraLex (2010) and PIC (2007): “The level and type (e.g. objectionable or not) of micro-organisms that can be present in raw materials, API starting mate-rials, intermediates or APIs. Bioburden should not be considered contamination unless the levels have been exceeded or defined objectionable organisms have been detected.
”The bioburden test in general is often seen as the test which is performed on the bulk solution used for parenteral products, either manufactured aseptically or terminally sterilized. The methods used are membrane filtration, plate count method or MPN according to Ph. Eur. 2.6.12 or USP . By defini-tion these methods are regarded as validated by the pharmacopeia. Whenever a different method is applied, it is advisable to validate this method according to Ph. Eur. 5.1.6., USP or PDA TR No. 33 (2013) and to show equivalence to the pharmacopeial method. Such validations for alternative or rapid microbiological methods (RMM) are available and are performed either by the supplier or some pharmaceutical companies.
However, each company that applies its own RMM should validate the method for its intended pur-pose. Since several applications are available on the market, a number of RMMs are presented in the following paragraph that can be used for bioburden testing without the claim of being exhaustive.
One of the basic questions when evaluating a RMM for bioburden testing is the limit of detection needed. An acceptance criterion of 10 cfu/100 ml is often used for the bioburden for parenteral products (EMEA 1996), however, it can be higher, as in biotech APIs, and then other methods with a less sensitive limit of detection can be used.