Downstream Purification Process Development

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 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

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

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

Cell Culture Primary Recovery Purification

0.2 micron

Typical Clarification Monoclonal Centrifugation and Depth Filtration Production Viral Capture Low pH Viral Antibody Bioreactor Inactivation Chromatography Inactivation Purification Purification Process R P

Intermediate Polishing Virus Tangential DS DS Chromatography Chromatography Filtration Flow UF/DF Dispensing Storage

Unit Operations Influencing the Attributes UF/DF Polishing Bioreactor Production Production Inactivation Clarification Intermediate Drug Substance Critical Low pH Viral Viral Filtration Viral Detergent Viral Tangential Flow Tangential Inactivation and Drug Substance Substance Drug Chromatography Chromatography Chromatography Primary Recovery Primary Quality Attributes A Protein Capture Dispensing/ Freezing Dispensing/ Product Related Impurities Aggregates O     Fragments O 

Post Translational Mods. O  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*     

Chromatography Bulk Operations Filtration Operations

R P

Protein A Affinity Flocculation Viral Filtration* Cation Exchange Detergent Viral Inactivation* Depth Filtration Anion Exchange Low pH Viral Inactivation* Tangential Flow UF/DF Hydrophobic Interaction Heat Inactivation Single-pass TFF Mixed-mode PEGylation Membrane Adsorbers Hydroxyapatite Enzymatic Reactions Dye Affinity * = dedicated viral clearance

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 Protein A Low pH Intermediate Polishing Virus Inactivation Chromatography Inactivation Chromatography Chromatography 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

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 Protein A improved selectivity and increased cleanability (base stability). Chromatography

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

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 Intermediate • Possible virus clearance Chromatography

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

10/5/2017 Company Confidential © 2017 Eli Lilly and Company 10 Polishing Chromatography 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 Polishing • Very strong HCP and Aggregate reduction Chromatography • Separation potential for truncated and misfolded product-related impurities • Possible virus clearance (works best with more hydrophilic proteins) Hydroxyapatite • Resin beads composed of crystalline Ca5(PO4)3(OH) • Strong Aggregate separation – but limited resin lifetime 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

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 Flocculation removal (DNA, HCP), and can lead to a simplified downstream process. A cleaner feedstream can allow Protein A to perform at higher level • 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 Chromatography”. Journal of Chromatography 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

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

Cell Culture Primary Recovery Purification

0.2 micron

Intermediate Polishing Virus Tangential DS DS Chromatography Chromatography Filtration Flow UF/DF Dispensing Storage