Controlling the quality of raw materials used in cell culture-based biotech manufacturing processes is a particularly challenging and critical task, because unlike traditional, small molecule manufacturing, an adventitious agent contamination event or other serious quality deviation has the potential to cause significant disruption to the manufacturing process and availability of the product (1).
The key to effective raw material control lies in developing a strategy that relies on thorough understanding of the role of raw materials in the manufacturing process as well as testing and supplier assessments.
Raw materials used in cell culture processes can be very diverse, ranging from inorganic salts to complex components like soy hydrolysates, animal-derived peptones or serum. Other raw materials commonly used in cell culture processes include carbohydrates, vitamins, trace elements, recombinant proteins such as insulin, defoaming agents, amino acids or nucleotides, among others. Examples of molecules manufactured by such processes are monoclonal antibodies or bifunctional fusion proteins.
Raw materials require clear control strategies in order to assure their quality. Therefore, the strategies presented herein can also be applied to other processes such as bacterial fermentation or chemical synthesis. As an example, Vitamin B12 (also called Cyanocobalamin) is used to illustrate the development of a raw material control strategy. Its role in mammalian cell culture processes is to serve as a cofactor for DNA synthesis in the cell, thus making it an essential material for any such process.
Vitamin B12 is mostly manufactured by bacterial fermentation, a well-known process applied for many years. Vitamin B12 is readily available on the global market, so continuous supplies are not an issue. It is possible to purchase directly from manufacturers which guarantees a good traceability of the material.
Definition of Raw Materials
The first step in setting a control strategy involves defining the raw materials in question. Due to the diversity of raw materials and also due to many other materials being used in the manufacture of biologics, it is important to be clear which material definition is applied for developing quality control strategies. In most cases, biologics are manufactured for distribution on the global market, therefore, the raw material definition of ICH Q7 (2) applies.
Elements of a Control Strategy For Raw Materials
Once the definition is set, a brainstorming exercise can be used to chart out the specific elements needed for a control strategy. The most important element is knowing why the material is being utilized, thereby understanding its role in the cell culture process. By this, the critical material attributes of a given raw material which may influence cell growth, and also product quality, are defined. [Note: The sidebar “Elements of a Raw Material Control Strategy” on p. 26 of the full PDA Letter describes additional elements of the brainstorming exercise.]
During this exercise, supplier qualification questionnaires—used to evaluate the quality system of the manufacturer and the quality of the raw material—are distributed, material for in-use/analytical testing is ordered, and if deemed necessary, audits may be performed at selected manufacturers based on the evaluation of the questionnaires and testing results.
Using the example raw material, the evaluation of materials from different Vitamin B12 providers consists of:
- Performing comparative small-scale cell culture with monitoring of the cell growth characteristics (“in-use testing”),
- Conducting a comparative assessment of whether there are differences in the results of analytical testing between manufacturers, and
- Testing additional to pharmacopoeial and/or manufacturer test methods may be needed due to special requirements of the cell culture process under assessment
When a criticality assessment is performed for all raw materials of a mammalian cell culture process, the outcome will most likely be that the criticality of Vitamin B12 will be considered “lower” compared to, say, serum components or recombinant proteins, but “higher” compared to buffers/salts used in the cell culture process.
Defining the Supplier Qualification Process
To optimize the supplier qualification process, this risk evaluation result can be used to identify items critical to raw material quality. Proposed elements crucial to the criticality assessment can include the following:
- Origin/complexity risk: Raw materials of human/animal/recombinant origin or premixed media are more complex, equaling higher risk than less complex components.
- Product contact: Raw materials with direct contact to product present higher risks (e.g., media component vs. fermenter cleaning).
- Impact on drug substance process: Raw materials used during cell culture with other functionality than cell growth promotion (e.g., antifoam agent for cell protection) present higher risk than those needed for cell growth.
These elements may then serve to refine the supplier qualification process, depending on the identified material risk. Table 1 shows an example of a material risk assessment for the general components of a mammalian cell culture process. A material cannot be rated with a category of “low” risk as a “yes” in a Product Contact category leads to a medium criticality material. This is due to the unique nature of a cell culture process. All components contribute and are in some way critical to ensure the proper manufacture of a biologic. As a next step, the elements of a manufacturer/supplier qualification for a raw material can be set up per criticality of material. Table 2 presents how medium and high criticality raw materials for cell culture processes could be assessed during the supplier qualification exercise.
|Table 1 General Example of a Material Risk Assessment |
|Definition: 1y=medium, 2-3y=high criticality (product contact risk = y prevails) |
|Criteria/Material ||Origin/complexity ||Product Contact ||Impact on DS process ||Risk |
|Inorganic salts/buffer components ||n ||y ||y/n ||Medium/High |
|Defoaming agent ||n ||y ||y ||High |
|Carbohydrate/energy source ||n ||y ||n ||Medium |
|Trace elements ||n ||y ||n ||Medium |
|Amino acids ||n ||y ||n ||Medium |
|Vitamins, Antioxidants ||n ||y ||n ||Medium |
|Nucleotides ||n ||y ||n ||Medium |
|Recombinant proteins ||y ||y ||n ||High |
|Serum or serum components ||y ||y ||n ||High |
|Hydrolysates/Peptones ||y ||y ||y ||High |
|Table 2 Supplier Qualification Elements Identified per Raw Material Criticality Definition |
|Manufacturer/Supplier Qualification for a Raw Material |
Define supplier qualification requirements for risk categories
|Medium Risk ||Provide CoA, specifications, test methods, MSDS |
Logistics, packaging, labeling
Supplier risk assessment based on questionnaires/supplier documentation
Change notification agreement
Testing of separate sample lots
|High Risk ||+ Process flow diagram, origin of materials used during manufacture, assess
risk of cross-contamination |
+ On-site audit
+ Confidentiality agreement, if needed
+ Contract, if needed
Raw Material Testing
Raw material testing is an essential part of a raw material control strategy. For testing, specifications need to be set up per raw material in order to evaluate the quality provided by the selected supplier. If this regime is applied to the Vitamin B12 example, the test list would entail testing as per pharmacopoeias, in-use testing, complete retesting of supplier CoA tests, and additional tests if required for a particular purpose.
The raw material testing regime mentioned above is outlined for materials to be used for market manufacture of a biological molecule. During development, a risk-based matrix for control is highly recommended in order to accommodate the phase of development. Minimal requirements for raw material control during development are defined in PDA Technical Report No. 56 (3). A tabular presentation can be found in Table 3. One element which requires a lot of time and effort during raw material supplier qualification is raw material supplier/manufacturer audits. Raw material suppliers are faced with multiple requests for audits by industry, and therefore, the Rx-360 initiative (4) was put in place as an option for audit information sharing.
|Table 3 Risk-based Matrix for Raw Material Control During Development |
|Note: Minimal requirements as per TR-56 |
|Development Phase ||Analytics ||Supplier |
(cell line/process development)
+ In-use testing
+ Document lots used
|Documentation of sources || |
+ Use well known and accepted suppliers, if available
+ Get multiple lots, if feasible
|Phase I/II || |
+ Identity testing
+ Method Development & Qualification
+ Supplier risk assessment (questionnaires)
+ On-site audit for high risk materials
+ Evaluate availability options
+ Logistics, packaging, labeling
|Phase III/ Validation || |
+ Full testing (CoA & in-house tests)
+ In-house stability
+ Method validation
+ On-site audits
+ Monitoring of approved Raw Materials
|+ Prepare supply/QA agreement (incl. change notification) |
(Annual Product Review)
|+ Check suitability of specifications for purpose ||+ Frequency of on-site audits based on experience ||+ Supplier monitoring |
Special Cases Need Special Attention
Raw materials used in the “pre-GMP” phase during establishment of the cell substrate are a concern to authorities, as they present a risk of contamination with adventitious agents. Whereas ICH Q7 defines the start of GMP activities with “Establishment of the Working Cell Bank (WCB),” the updated EU Annex 2 requires the start of GMP activities at Master Cell Bank establishment level (5). This means that qualified raw materials should be used for these activities. As described in Table 3, at minimum, CoA/CoO/Transmissible spongiform encephalopathy (TSE) information are mandatory to be available for each raw material. In TR-56, Appendix 6.0 deals with “Quality Systems Applicable To Cell Culture Development.” The EU even goes one step back into cell line development and requires that for stages prior to the cell bank generation, “...documentation should be available to support traceability including issues related to components used during development with potential impact on product safety...” (from EU Annex 2 No. 41). These requirements are not new, as the ICH Guideline Q5D (6) section 2.1.2 also has separate paragraphs applying to cell substrate and the cell line used for transfection. It is highly recommended to obtain parental cell lines from trusted sources with appropriate documentation and source any raw materials used during cell line development from GMP-qualified raw materials as much as possible. In addition, keeping good track of raw material documentation ensures data integrity. Only then, it can be assured at the time of filing a biologics application that the safety risk brought by raw materials of all development stages—even those prior to GMP—can be appropriately assessed, rather than starting with worst case considerations.
The use of raw materials of animal/human origin in a cell culture process bears a significant risk for contamination with adventitious agents. These typical raw materials are: fetal calf serum/fetal bovine serum, bovine serum albumin (BSA) and human serum albumin (HSA). Guidance is available from either ICH or WHO, as well as the ICH regions: the United States, European Union and Japan, which describe what testing is required to assure safe use of such raw materials. As an example, use of HSA requires fulfilling a lot of provisions in all regions, so it is highly advisable to source globally registered and authority-released products only. Look-back procedures in case of HSA recalls need to be in place; the timeline for this is very long (30 years), so HSA manufacturers have to ensure in their SOPs that these requirements are met. The use of any other human-origin material which is not covered by licenses bears an even higher risk as there is no authority oversight, and the “end user” will be required to comply with provisions very similar to those for HSA which can cause significant additional documentary needs, testing and risk assessments. The use of raw materials of animal/human origin in cell culture processes is difficult and may necessitate additional measures to assure the safety of the product manufactured. It is also highly recommended to apply the relevant authority guidance, as well as any supplier qualification provisions, to animal/human–origin materials used in the “pre-GMP” phase. A sourcing and QA agreement should be in place from the start in order to mitigate any adventitious agents risk.
Raw material quality control strategies are a very important element to ensure the safety, quality and efficacy of the biological molecule to be manufactured. Several aspects have to be considered to achieve this:
- Understanding the role of the raw materials in the process
- Developing testing strategies to ensure raw material quality
- Evaluating whether specific tests are needed for the particular raw material in order to ensure consistent material quality
- Setting up material/supplier risk assessments in the context of a supplier qualification exercise
- Having a supplier qualification system in place which assesses all risks of a raw material: origin risk (safety), supply risk (availability, back-up options, audits, contractual agreements) and quality risk (testing, evaluation of lot-to-lot consistency).
If implemented diligently, raw material quality control strategies help to assure the quality of the product, and contribute to mitigate the risk of adventitious agent contamination.
- Aranha, H. Virus Safety of Biopharmaceuticals. Contract Pharma 13 (2011) tinyurl.com/kpom6bq
- ICH Harmonised Tripartite Guideline: Good Manufacturing Practice Guide For Active Pharmaceutical Ingredients Q7 tinyurl.com/3u5st59
- Eylath, A., et al. PDA Technical Report No. 56: Application of Phase-Appropriate Quality Systems and CGMP to the Development of Therapeutic Protein Drug Substance. Bethesda, MD: Parenteral Drug Association, 2012.
- Rx-360. “About Rx-360.” rx-360.org. tinyurl.com/lvb7n72 (accessed Oct. 10, 2014)
- EudraLex The Rules Governing Medicinal Products in the European Union Volume 4 EU guidelines for Good Manufacturing Practice Annex 2 tinyurl.com/m73bvsw
- ICH Harmonised Tripartite Guideline Derivation And Characterisation Of Cell Substrates Used For Production Of Biotechnological/Biological Products Q5D tinyurl.com/3pc7hmn
About the Author
Annemarie Möritz, PhD, has over 20 years of experience both in the pharmaceutical and IVD industry, and is a member of the PDA BioAB.