To gain perspective on the implementation of quality by design (QbD) and process analytical technology (PAT) in biopharmaceutical processing, BioPharm International spoke with Clinton Weber, associate director of bioprocess sciences, CMC Biologics Organization; Henrik Johanning, director QAtor; Nathan L. McKnight, PhD, principal engineer, late stage cell culture, BioProcess Development, Genentech; Anurag Rathore, professor, Indian Institute of Technology (IIT) Delhi; Frederic Girard, CEO, Spinnovation Biologics; Thomas J. Vanden Boom, PhD, vice-president, global biologics research, development and manufacturing operations, Hospira.
BioPharm: In implementing QbD, what would you identify as the critical quality attributes (CQAs) in a typical upstream bioprocess using cell-culture?
Vanden Boom (Hospira): Generally, upstream critical quality attributes for a cell-culture manufacturing process are limited to the adventitious agent and bioburden testing of the cell-culture harvest material. This also holds for biosimilar products.
McKnight (Genentech): CQAs are defined for the product, not identified as part of upstream or downstream portions of the manufacturing process. There are key performance indicators (KPIs) that are defined for upstream steps, including culture productivity (i.e., titer), cell growth, and viability. While KPIs may be correlated with CQA results, KPIs are not themselves product quality attributes. However, particular CQAs may be generated or modified during specific upstream or downstream steps. Protein glycosylation, for example, is generally determined during cell culture and minimally altered in downstream unit operations (at least for uncharged glycosylation species). Using this definition of CQAs, those CQAs observed to be potentially impacted during cell-culture steps include product attributes contained within the protein glycan distribution (e.g., afucosylated glycan), charge-variant distribution (e.g., glycated, deamidated forms), and to a lesser extent, molecular-size distribution (e.g., dimer or aggregate forms).
It should be noted that knowledge of an association between cell-culture steps with certain product quality attributes is not a result of implementing a QbD approach but, rather, a result of knowledge gained through the basic scientific and engineering endeavors that should be elements of developing a bioprocess.
Rathore (IIT Delhi): In the past, most CQAs could not be measured directly in the fermenter broth due to the interference from the numerous components present in the broth. As a result of major advancements in analytical science, direct measurements of CQAs are performed in bioprocessing today. These would be product-quality related parameters, including host-cell impurities (e.g., host-cell proteins, DNA), process-related (e.g., Protein A leachate), and product related (e.g., aggregate, basic variants, acidic variants, and glycoxylation pattern). Some of these CQAs, such as glycoxylation pattern for monoclonal antibody (mAb) products, are primarily impacted by the upstream process and are particularly important to monitor during process development.
Weber (CMC Biologics): The goal of a QbD approach is to develop additional knowledge of the impact of upstream process unit operation performance on the final purified product quality. The most likely or desired outcome is to develop a quantifiable correlation between upstream process outputs, such as cell viability or viable cell density (VCD), and product attributes, such as glycosylation. This approach can lead in the determination of CQAs for the cell production bioreactor unit operation. Upstream outputs classified as CQAs have proven to be controversial, because the bioreactor is so far upstream from the final product. However, if a strong correlation can be established between VCD/viability and other CQAs or final product specifications, this can be an appropriate approach.
Girard (Spinnovation): The underlying concept for QbD of an upstream bioprocess is that the desired quality of the biological or biopharmaceutical product is assured every time. CQAs vary for each cell line depending on the nature of the bioprocess, with typical critical qualities such as metabolites and contaminants. CQAs usually include properties that affect product quality and eventually overall performance of the bioprocess. CQAs are typically also release tests, although they don't have to be as there are no real release tests as such in upstream processing.
The variability and complexities associated with the upstream biological process make QbD a complex process, one that relies on defining operation specific critical process parameters (CPPs). CPPs are those likely to impact on the quality of a product or intermediate. For biological products, process control can be difficult to define and implement. O2 pressure, catalyst concentration, and pH are examples of critical parameters. It is important to note that mAbs are currently the leading area of biopharmaceutical research. One of the key parameters to monitor in the implementation of QbD in mAb production is the glycosylation process during formulation. Glycosylation is one of the overriding contributors to mAb heterogeneity and has significant implications for the function of the antibody in vivo and immunogenicity. This means that glycosylation has been isolated as a critical parameter to follow during mAb manufacture. QbD for mAb development with specific glycosylation patterns enables researchers to optimize manufacturing and clinical efficiency.
Johanning (QAtor): The QbD concept works its way back so to speak from the patient to product to process and ultimately to the facility. A risk assessment will outline the risk profile in and between each area.
The starting point for the risk assessment is R&D, which upstream has determined the CQAs on the product. CQAs are often product specifications, including eventual GMP requirements (if GMP is used as part of the biopharmaceutical process, which is often the case in multinational pharma companies in order to ensure a fast-track initiative from R&D to license to operate and market). The risk assessment includes a review and assessment of the products CQAs when manufactured on specific equipment.
BioPharm: In implementing QbD, what would you identify as the critical process parameters (CPPs) in a typical upstream bioprocess using cell culture?
McKnight (Genentech): Among cell-culture parameters, culture pH is typically the most difficult to control relative to its impact on cell-culture performance and product quality. This is generally the case for both mAbs and other products.
Johanning (QAtor): Common CQAs in an upstream bioprocess (i.e., bio formulation process) using cell-culture include sterility and bioactivity.
Rathore (IIT Delhi): Typical CPPs for a fermentation step would be pH, sparge rate, agitation rate, and temperature. These are typically easy to control. Issues arise when one goes to volumes greater than 1000 L and when it increasingly becomes more difficult to strip off the CO2 generated by the cells and in ensuring uniform supply of O2 and other critical nutrients to the cells. Another set of challenges comes from raw materials that are complex and not well characterized (such as yeastolates) as these can result in significant variation in the CQAs from lot to lot. For mAbs, besides the afore-mentioned factors, concentration of critical nutrients has been known to affect the glycoxylation pattern of the product, thereby impacting product efficacy.
Vanden Boom (Hospira): Parameters such as temperature, pH, osmolality, and dissolved oxygen have the potential to impact the CQAs of mAbs and other mammalian cell-culture derived products. However, with current bioreactor engineering controls, these parameters may be tightly and confidently controlled within the design space of the manufacturing process permitting these parameters to be downgraded from a CPP to a key process parameter (or other lower parameter designation used by different drug sponsors).
Weber (CMC Biologics): For an upstream process, the process of expanding the cells is the primary purpose until the culture goes into the production bioreactor. Because the majority of product produced is in the production bioreactor, the expansion process is considered to have minimal impact on the final product. Additionally, most products have shown the ability to recover from suboptimal conditions during expansion without serious product quality impact. Therefore, for a typical upstream process, CPPs are identified at the production bioreactor stage. CPPs for the production bioreactor may include seed density, temperature, and process duration. Initial seed density can impact the overall growth profile and viability. Temperature is critical in maintaining viability and may impact the quality of product being produced by the cells. Process duration will impact the viability of the culture at harvest, which can be tied to product quality.
Among the most difficult parameters to control in an upstream process is CO2 concentration. The difficulty of controlling this parameter depends on the complexity of the aeration control strategy and availability of dissolved CO2 probes. Though there can be typically broad ranges of acceptable CO2 during production, very high CO2 concentrations can impact the product quality. A balance needs to be maintained between lowering CO2 and maintaining pH at a set point. Furthermore, these conclusions seem to be supportive of most cell-culture processes, not just mAb production.