Process Removal of Impurities in Biotech Products

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1 Process Removal of Impurities in Biotech Products CASSS Midwest Regional Forum October 5, 2017 Warren R. Emery Sr. Research Scientist Bioproduct R&D, Eli Lilly and Company

2 Pharmaceutical Process Development PHARMACEUTICAL DEVELOPMENT The aim of pharmaceutical development is to design a quality product and its manufacturing process to consistently deliver the intended performance of the product. The information and knowledge gained from pharmaceutical development studies and manufacturing experience provide scientific understanding to support the establishment of the design space*, specifications, and manufacturing controls. ICH Q8 R2 The downstream purification process must be designed to control a wide variety of critical quality attributes, including impurities and contaminants Company Confidential 2017 Eli Lilly and Company 2

3 Impurities Process-related impurities encompass those that are derived from the manufacturing process, i.e., cell substrates (e.g., host cell proteins, host cell DNA), cell culture (e.g., inducers, antibiotics, or media components), or downstream processing. HCP, DNA Detergent, Flocculant, Leached Protein A, processing enzymes, PEG reagents Product-related impurities (e.g., precursors, certain degradation products) are molecular variants arising during manufacture and/or storage that do not have properties comparable to those of the desired product with respect to activity, efficacy, and safety. Aggregates Fragments Post translational modifications, sequence variants ICH Q6B Company Confidential 2017 Eli Lilly and Company 3

4 Contaminants Contaminants in a product include all adventitiously introduced materials not intended to be part of the manufacturing process, such as chemical and biochemical materials (e.g., microbial proteases) and/or microbial species. POTENTIAL SOURCES OF VIRUS CONTAMINATION Viral contamination of biotechnology products may arise from the original source of the cell lines or from adventitious introduction of virus during production processes. A. Viruses That Could Occur in the Master Cell Bank (MCB) Cells may have latent or persistent virus infection (e.g., herpesvirus) or endogenous retrovirus which may be transmitted vertically from one cell generation to the next, since the viral genome persists within the cell. B. Adventitious Viruses That Could Be Introduced during Production Adventitious viruses can be introduced into the final product by several routes including, but not limited to, the following: 1) the use of contaminated biological reagents such as animal serum components; 2) the use of a virus for the induction of expression of specific genes encoding a desired protein; 3) the use of a contaminated reagent, such as a monoclonal antibody affinity column; 4) the use of a contaminated excipient during formulation; 5) contamination during cell and medium handling. ICH Q6B, ICH Q5A Company Confidential 2017 Eli Lilly and Company 4

5 Downstream Purification - Overview Cell Culture Primary Recovery Purification 0.2 micron Typical Monoclonal Antibody Purification Process Production Bioreactor Clarification Centrifugation and Depth Filtration Purification Viral Inactivation Capture Low ph Viral Inactivation R P F Intermediate Polishing Virus Filtration Tangential Flow UF/DF DS Dispensing DS Storage Company Confidential 2017 Eli Lilly and Company 5

6 Control Points Matrix Drug Substance Critical Quality Attributes Product Related Impurities Unit Operations Influencing the Attributes Production Bioreactor Primary Recovery Detergent Viral Inactivation Protein A Capture Low ph Viral Inactivation and Clarification Intermediate Polishing Viral Filtration Tangential Flow UF/DF Aggregates O Fragments O Post Translational Mods. O Drug Substance Dispensing/ Freezing Process Related Impurities Residual DNA O Residual Host Cell Proteins O Residual Protein A O Residual Detergent O Media Components O Contaminants Microbial Safety Viral Safety O* Company Confidential 2017 Eli Lilly and Company 6

7 Purification Platform Toolbox Approach Bulk Operations Filtration Operations R P F Protein A Affinity Cation Exchange Anion Exchange Hydrophobic Interaction Mixed-mode Hydroxyapatite Dye Affinity Flocculation Detergent Viral Inactivation* Low ph Viral Inactivation* Heat Inactivation PEGylation Enzymatic Reactions Viral Filtration* Depth Filtration Tangential Flow UF/DF Single-pass TFF Membrane Adsorbers * = dedicated viral clearance Company Confidential 2017 Eli Lilly and Company 7

Viral Safety Mission: To design/develop downstream purification processes with robust and consistent viral clearance capacity in support of clinical trials and commercialization = Dedicated viral

8 Viral Safety Mission: To design/develop downstream purification processes with robust and consistent viral clearance capacity in support of clinical trials and commercialization = Dedicated viral clearance unit operations Detergent Inactivation Protein A Low ph Inactivation Intermediate Polishing Virus Filtration Dedicated viral clearance unit operations ensure orthogonal and robust safety margins for retrovirus broad platform applicability Other unit operations may provide additional clearance capacity these are more process specific than dedicated operations AEX, Protein A, Heat Inactivation - CEX, HIC, Mixed Mode Company Confidential 2017 Eli Lilly and Company 8

9 Capture - Protein A Affinity Protein A: 42 kda protein found in the cell wall of the bacteria Staphylococcus aureus. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region of human VH3 family. A wide variety of Protein A resins are available, including engineered forms with improved selectivity and increased cleanability (base stability). Protein A PROS Expensive resin, but highly selective affinity mode of chromatography (contributes to ROI) Robust (multi-log) reduction of DNA, HCP, media components, detergent, etc. Moderate viral clearance capability CONS Protein A leaching that must be controlled downstream Company Confidential 2017 Eli Lilly and Company 9

10 Intermediate Goal: To reduce and control multiple process and product related impurities. Cation Exchange (typically bind/elute) Manufacturing friendly high load ratio, simple buffers Strong HCP, Aggregate, DNA reduction, possible product-related impurities Possible virus clearance Intermediate Anion Exchange (b/e, flowthrough, membrane) Manufacturing friendly (very) high load ratio, simple buffers Predictable and generally robust virus clearance Strong DNA and modest HCP and Aggregate reduction, possible product-related impurities Mixed Mode (IEX/hydrophobic) (b/e, flowthrough) Manufacturing friendly (very) high load ratio, simple buffers Alternative selectivity to straight CEX or AEX opportunities for optimization Possible virus clearance Company Confidential 2017 Eli Lilly and Company 10

Polishing Goal: To provide an orthogonal mode of separation and serve as the final control point for multiple process and product related impurities.

11 Polishing Goal: To provide an orthogonal mode of separation and serve as the final control point for multiple process and product related impurities. Hydrophobic Interaction Less manufacturing friendly lower load ratio, heavy usage of kosmotropic salts Very strong HCP and Aggregate reduction Separation potential for truncated and misfolded product-related impurities Possible virus clearance (works best with more hydrophilic proteins) Hydroxyapatite Resin beads composed of crystalline Ca 5 (PO 4 ) 3 (OH) Strong Aggregate separation but limited resin lifetime Polishing Dye Affinity (Cibacron blue, etc.) Textile dyes dye structure consists of a chromophore, linked to a reactive group, with sulfonic acid groups tend to interact with binding sites on proteins Unique selectivity for many proteins especially enzymes Company Confidential 2017 Eli Lilly and Company 11

12 Case Study Up-Front HCP Control Problem: DNA and HCP reduction across Protein A are good, but can the affinity column perform even better? Flocculation at the end of cell culture (chitosan, pdadmac) can significantly improve performance of primary recovery. Newer flocculation techniques can also play a significant role in impurity removal (DNA, HCP), and can lead to a simplified downstream process. A cleaner feedstream can allow Protein A to perform at higher level Flocculation Proprietary flocculation technique by Gagnon group at Bioprocess Institute, Singapore New stimulus reactive polymer from Merck Millipore (evaluated at Eli Lilly) Nian, R., et. al., 2016, Advance Chromatin Extraction Improves Capture Performance of Protein A Affinity. Journal of A Kang, Y., et. al., 2013, Development of a Novel and Efficient Cell Culture Flocculation Process Using a Stimulus Responsive Polymer to Streamline Antibody Purification Processes. Biotechnology and Bioengineering Company Confidential 2017 Eli Lilly and Company 12

13 Case Study 2-for-1 Inactivation Problem: Some product molecules may be susceptible to enzymatic degradation by proteases that are expressed by mammalian cell culture HCPs with enzymatic activity can impact the stability of some bioproducts if so, they must be controlled by the purification process Enzymatic activity may be present at HCP levels below our ability to detect Heat Inactivation Heat inactivation of enzymes takes advantage of differences in thermal stability between the product molecule and the enzymatic HCP Some viruses are also susceptible to heat treatment to achieve inactivation May be performed in batch at lower temp/longer time or continuously by HTST Lambooy, P., et. al., 2008, Heat Inactivation of Protease During Downstream Processing of a Fusion Protein Enables Purification of a Stable Bulk Drug Substance. Recovery Conference Bailey, M., et. al., 2007, Evaluation of Microfluidics Reactor Technology on the Kinetics of Virus Inactivation. Biotechnology and Bioengineering Company Confidential 2017 Eli Lilly and Company 13

14 Downstream Purification - Overview Cell Culture Primary Recovery Purification 0.2 micron Typical Monoclonal Antibody Purification Process Production Bioreactor Clarification Centrifugation and Depth Filtration Purification Viral Inactivation Capture Low ph Viral Inactivation R P F Intermediate Polishing Virus Filtration Tangential Flow UF/DF DS Dispensing DS Storage Company Confidential 2017 Eli Lilly and Company 14

Modular Retrovirus Clearance in Support of Clinical Development

Modular Retrovirus Clearance in Support of Clinical Development Bio-product Research & Development, Eli Lilly and Company CMC Strategy Forum, Europe, 2018 D. Chen/ M. Murphy General Expectations for Viral