Navigating the Biopharmaceutical Regulatory Pathway: Replacing Undened Raw Materials with Chemically Dened Substitutes

BD Biosciences Advanced Bioprocessing, Cockeysville, MD

ABSTRACT
Traditionally, manufacturers of biopharmaceuticals cultured cells in cell culture media supplemented with bovine serum. However, with the discovery of the infectious agent responsible for bovine spongiform encephalopathy (BSE), prions, increased safety concerns and country regulations have led them away from serum-based media and toward chemically dened (CD), animal-free supplements. At the same time, manufacturers are reluctant to abandon a working process and incur the administrative burden of a regulatory re-le. This white paper presents an analysis of a next-generation media supplement, BD Recharge without Glucose and L-Glutamine. BD Recharge was specically designed as a chemically dened supplement that offers protein productivity and quality comparable to supplementation with a yeast extract peptone, yet helps to minimize the regulatory burden of a changed process.

THE EVOLUTION OF MEDIA SUPPLEMENTATION

Until the late 1980s, manufacturers of biopharmaceutical products cultured their cells in fetal bovine serum, despite lotto-lot variability (quantitatively and qualitatively) and supply issues. With the discovery of prions in the late 1980s and
Figure 1. The evolution of media supplementation.
Cells cultured in bovine serum In late 1980s, BSE discovered Increasing movement away from serum-based media

increasing country regulations, manufacturers began to explore alternatives to serum-based media. They began to reduce risk by moving to peptones of animal origin, and then to animal-free peptones, such as those based in yeast or soy.

Common usage of serum alternatives, including peptones (some of animal-origin) to boost protein production Peptone lot-to-lot variability problematic for yield Increasing movement away from animal origin components

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Early adoption of animal-origin peptones as serum alternatives

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First generation of chemically dened media supplements Movement toward chemically dened animal-free components

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Although both serum and peptone supplements are rened from their origins, they share the disadvantage of being undened. No one can say exactly what is in them, and their composition can vary from lot-to-lot. Recently, interest has intensied in mitigating risk by switching from less dened supplements to those in which all components are entirely

chemically dened (CD), animal-free, and protein free (Figure 2). By using dened sources, CD supplements yield more reliable performance and improved lot-to-lot consistency. The challenge for CD supplements is to match current supplementation methods in productivity and nal product protein quality.

Figure 2. Risk from animal-origin ancillary materials used in culture media.

HIGH LOW

SERUM
REFINED

AO PEPTONES

AF PEPTONES

CHEMICALLY DEFINED
DEFINED

Competing pressures have thus put biopharmaceutical manufacturers in a dilemma (Figure 3). On the one hand, migrating to CD supplements benets manufacturers by providing a consistent, predictable supply of raw materials, resulting in a lower risk of contamination and lower batch failure rates. CD supplements can also improve lot-to-lot consistency, increasing condence in process performance/capability and mitigating risk associated with undened (and especially animal-origin) components. Regulatory agencies generally support this move away from animal-based components because it reduces exposure to impurities and adventitious agents.

On the other hand, once a regulatory authority has approved a manufacturing process, manufacturers have an incentive to maintain the status quo. Changes in process increase their regulatory burden. In addition, any changes to inputs, equipment, or process must be assessed for impact on the nal biotherapeutic. Manufacturers must develop explicit comparability protocols to assess possible effects on the purity, efcacy, and quality of the product, in terms of both safety and effectiveness. They may have to qualify the new supplement, revalidate new process steps, or even conduct new clinical trials.

Figure 3. The manufacturers dilemma: Is a process change justied?

Improved Consistency Lower batch failure rates Higher capacity utilization Yields Upstream and downstream yields Risk Reduction Elimination of undened and/or animal origin components Lower risk of contamination

VS

Switching Costs Qualication / Validation CLINICAL TRIALS

BD RECHARGE

BD Recharge is a chemically dened media supplement recommended for use with Chinese hamster ovary (CHO) cells. It was designed to not only match the increase in protein yield provided by BD Biosciences yeast extract-based hydrolysates but to reduce biopharmaceutical manufacturers regulatory burden. Scientists from BD Biosciences created this next-generation CD supplement by reverse-engineering yeast extract peptones

in a step-wise experimental paradigm. They began with a proprietary fractionation process to separate the constituents of yeast extract, an animal-free peptone. Then, they used iterative Design of Experiments (DOE) methodology (Figure 4) to identify and optimize the key bioactive and nutritional components in the formulation. These key components of the yeast extract peptone were sourced and combined into a single powder supplement.

Figure 4. Optimization of BD Recharge through an iterative DOE methodology. A.

B.

Individual components identied through fractionation were subjected to DOE to identify and optimize the nal formulation. In Panel A, six different components (C1 C6) were optimized. Each component was tested at three different concentration levels (low, medium, and high, relative to that component). Each dot represents the specic component concentration that was utilized in the mixture. Forty independent mixtures were generated and then tested in the CHO biological assay system. In this assay, many parameters were measured including protein yield, viable cell density, and viability. Panel B shows the protein yield of the 40 mixtures

in this DOE. The mixtures with an asterisk (3, 39, 40) had the highest production yields and were further explored. The result is a complete, chemically dened supplement that includes only the key bioactive and nutritional constituents of the yeast extract peptone. And, because all components in BD Recharge are present in the existing yeast extract peptone, no new chemicals or entities are being added to the process stream. This is demonstrated in a sample chromatogram overlay of one of several constituent groups (Figure 5), which shows that all peaks in BD Recharge are also present in the yeast extract peptone.

Figure 5. Chromatogram mirror overlay. A yeast extract peptone (black / bottom) and BD Recharge (orange / top) were subjected to chromatographic analysis of one of several constituent groups. Although the yeast extract peptone showed some unique peaks of its own, all peaks in BD Recharge were also found in the yeast extract peptone. Chromatographic comparisons of other constituent groups showed similar results.

CHROMATOGRAM OVERLAY
Orange = BD Recharge Black = Yeast

PROTEIN PRODUCTIVITY AND EQUIVALENCE

Was this unusual design strategy effective? Figure 6 shows the protein yield of a CHO cell line expressing a monoclonal antibody that has been grown in media supplemented with either a yeast extract peptone or with BD Recharge. BD Recharge

achieved equivalent protein yield to yeast extract peptone, with lower variability. Both demonstrated signicantly higher yields than the media-only control.

Figure 6. Protein yield: BD Recharge vs. yeast extract supplementation. CHO cells expressing a monoclonal antibody were grown in media with no supplementation (media-only control) or supplementation with yeast extract peptone or BD Recharge. Results: Protein production from cells grown in media supplemented with BD Recharge was greater than from cells grown in media supplemented with yeast extract peptone and signicantly greater than the media-only control.

Of course, by denition, one would expect a media supplement to increase protein production. Equally important when changing a process is that no changes in the recombinant protein therapeutic are introduced. To clear an important regulatory hurdle, the protein produced by the new process must be proven comparable to the original protein. Because the active components of BD Recharge CD supplement are the same as the yeast extract peptone it was based on, the proteins produced by the two processes should be biochemically identical.

One measure of protein comparability is its charge variance. When passed through an ion exchange column, the proteins are separated and eluted based on their overall charge. Figure 7 shows the charge proles of monoclonal antibody produced using either yeast extract or BD Recharge as the cell culture media supplement. The two are virtually identical. Although charge variance is only one measure of comparability, it is an important indicator of overall protein quality, which can impact protein efcacy.

Figure 7. Protein quality: BD Recharge vs. yeast extract supplementation. Monoclonal antibodies produced by CHO cells grown in media supplemented with either BD Recharge or yeast extract were subjected to chromatographic analysis. Results: For all eight peaks, the charge variance of the antibodies produced with BD Recharge was comparable to those produced with yeast extract.

Customer testing of BD Recharge by a large, international biopharmaceutical company has shown similar comparability in ion exchange charged variants (measured by ion-exchange chromatography), aggregation (measured by size-exclusion chromatography), and protein integrity (measured by reversed phase chromatography). By these measures, only negligible

differences were found between the proteins produced in bioreactors with media that had been supplemented by yeast extract versus BD Recharge. Even N-terminal glycosylation patterns (measured by N-glycan analysis) were similar, despite their sensitivity to small changes in cell culture media or conditions.

REGULATORY IMPLICATIONS

Relatively small changes in a manufacturing process can alter the characteristics of a drug and thus affect safety and/or efcacy.1 ICH guideline Q5E (November 2003) species criteria for the comparability of biotechnical and biological products subject to changes in their manufacturing process. Biopharmaceutical manufacturers should be aware of three categories of product quality characteristics to be assessed for compatibility: Specic changes in the protein molecule being produced, including its physical, chemical, and biological characteristics. These include size, charge, and mass heterogeneity when native or denatured; reduced or not reduced; post-translational modications such as glycosylation; and potency and activity. Product-related impurities and degradants, often resulting from alterations in the protein molecule. These alterations include truncation, aggregation, precipitation, deamidation, oxidation, hydrolysis, photolysis, and disulde scrambling.

Process-related impurities and contaminants, often resulting from changes in the upstream material components. These include host cell proteins, host cell DNA, and media constituents; process additives such as antibiotics, enzymes, and antifoam; afnity ligands such as Protein A; and microbial contamination. In this case, BD Recharge introduces no new components into the upstream process. In addition, because all components in BD Recharge are less than 500 Daltons in molecular weight, they would routinely be removed during downstream processing. Figure 8 shows a range of typical methodologies used in comparability testing for monoclonal antibodies. To develop a comparability protocol, manufacturers should know enough about their molecule and process to select those attributes that are most likely to be affected by the process changes they are considering.

Figure 8. Typical comparability testing methods for monoclonal antibodies.2,3

Purity/Isoforms/Degradants HPLC/UPLC = SEC, RP, IEX, HIC Gel Electrophoresis = SDS-PAGE, IEF Capillary Electrophoresis = CZE, CLIF, CIEF, CSDS Conformation Circular Dichroism Differential Scanning Calorimetry Potency Binding Assays (Fab) Effector Assays (Fc) Quantity Protein Concentration

Identity/Appearance Peptide Mapping Oligosaccharide Mapping Color/Clarity/Visible and Subvisible Particulates Mass Characterization Mass Spectrometry Analytical Ultra Centrifugation Light Scattering Process Impurities/Contaminants HCP, DNA Media Components Afnity Ligand Leachate Bioburden Pyrogens

BD Recharge is unusual in having a relatively small change footprint. It contains no new components, and those that remain are chemically dened and animal free. Because BD Recharge provides only the essential components of the yeast extract peptone, the elimination of non-essential components delivers a predictable formulation with lower lot-to-lot variation. Finally, chromatography and charge variance proles conrm that a typical protein product is comparable. (Manufacturers will need to conrm this for their own product.) Given these attributesand assuming conrmation of protein comparabilityBD suggests that migration from yeast extract to BD Recharge for the manufacture of products that are already approved may qualify for the least burdensome regulatory path. In the US, changing to BD Recharge might be led with the FDA as a supplemental application for Changes Being Effected in 30 Days (CBE-30), which requires a 30-day review period before a product made using the change can be marketed.4 In the EU, changing to BD Recharge might be led with the EMA as a Type II Variation, an amendment to the marketing authorization with a standard review period of 60 days.5 In

both cases, the manufacturer must supply all necessary comparability data to demonstrate that the switch to BD Recharge does not affect their nal product quality. It is essential for the manufacturer to have an open partnership with the supplement supplier both before and during the approval process. Because each process is unique, the supplier must assist the manufacturer with technical support to ensure proper utilization of the supplement. What works in one system or process may not work in another. BD is available to provide technical support for proper formulation and use of BD Recharge in the test and implementation phase. In addition, BD regulatory experts understand the Chemistry, Manufacturing, and Controls (CMC) requirements and are ready to work with Biologics License Application (US) and Marketing Authorization Holder (EU) licensees to support the application process. In the case where the required product information for regulatory ling is proprietary or a trade secret, BD will work with the customer on a case-by-case basis to support the customer needs.

SUMMARY

Advances in the development of cell culture media are making chemically dened (CD) supplements an increasingly attractive alternative to serum or peptones. Although biopharmaceutical manufacturers are understandably reluctant to abandon a regulatory-approved process, switching from an undened supplement to CD can make both regulatory and business sense. BD Recharge without Glucose and L-Glutamine was presented as an example. Developed from, and containing only a subset of, the components of yeast extract, BD Recharge matches or exceeds yeast extract in protein production, with

less analytical variability. Moreover, proteins manufactured using BD Recharge are virtually indistinguishable on several measures from proteins manufactured using yeast extract. The lack of new or additional components in a CD supplement may ease the regulatory path to acceptance, as long as the manufacturer can demonstrate the comparability of the end product (protein). To maximize the chance of success, the CD supplement supplier must be willing and able to partner with the manufacturer during evaluation, acceptance, and regulatory re-le.

REFERENCES
1. Towns J & Webber K, Demonstrating comparability for well-characterized biotechnology products: Early phase, late phase, and post-approval. Bioprocess International, pp 32-43 (Feb 2008). 2. Lubiniecki A, et al., Comparability assessments of process and product changes made during development of two different monoclonal antibodies. Biologicals 39(1): 9-22 (2011). 3. Schenerman M, et al., Analysis and Structure Characterization of Monoclonal Antibodies; CMC Strategy Forum Report. Bioprocess International, pp 42-50 (Feb 2004). 4. US 21 CFR 601.12(c) and 314.70(g). 5. EC Directive No. 1234/2008.

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