Upload
others
View
9
Download
1
Embed Size (px)
Citation preview
Process Removal of Impurities in Biotech Products
Warren R. EmerySr. Research ScientistBioproduct R&D, Eli Lilly and Company
CASSS Midwest Regional ForumOctober 5, 2017
Pharmaceutical Process Development
Company Confidential © 2017 Eli Lilly and Company 210/5/2017
ICH Q8 R2
PHARMACEUTICAL DEVELOPMENT
The aim of pharmaceutical development is to design a quality product and itsmanufacturing process to consistently deliver the intended performance of theproduct. 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.
• The downstream purification process must be designed to control a wide variety of critical quality attributes, including impurities and contaminants
Impurities
Company Confidential © 2017 Eli Lilly and Company 3
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
10/5/2017
Contaminants
Company Confidential © 2017 Eli Lilly and Company 4
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 CONTAMINATIONViral 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 ProductionAdventitious 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 Q5A10/5/2017
Downstream Purification - Overview
Company Confidential © 2017 Eli Lilly and Company 5
Typical MonoclonalAntibody PurificationProcess
10/5/2017
F
RP
Production Bioreactor
ClarificationCentrifugation and Depth Filtration
0.2 micron
Viral Inactivation
Capture Chromatography
Low pH Viral Inactivation
Intermediate Chromatography
Polishing Chromatography
Virus Filtration
Tangential Flow UF/DF
DS Dispensing
DS Storage
Primary Recovery PurificationCell Culture
Purification
Control Points Matrix
Company Confidential © 2017 Eli Lilly and Company 6
Drug Substance Critical Quality Attributes
Unit Operations Influencing the Attributes
Prod
uctio
n
Bio
reac
tor
Prim
ary
Rec
over
y
Det
erge
nt V
iral
Inac
tivat
ion
Prot
ein
A C
aptu
re
Chr
omat
ogra
phy
Low
pH
Vira
l
Inac
tivat
ion
and
Cla
rific
atio
n
Inte
rmed
iate
Chr
omat
ogra
phy
Polis
hing
Chr
omat
ogra
phy
Vira
l Filt
ratio
n
Tang
entia
l Flo
w
UF/
DF
Dru
g Su
bsta
nce
Dis
pens
ing/
Fre
ezin
g
Product Related ImpuritiesAggregates O Fragments O
Post Translational Mods. O
Process Related ImpuritiesResidual DNA O
Residual Host Cell Proteins O
Residual Protein A O
Residual Detergent O
Media Components O
ContaminantsMicrobial Safety
Viral Safety O*
10/5/2017
Purification – Platform Toolbox Approach
Company Confidential © 2017 Eli Lilly and Company 7
Chromatography
Protein A AffinityCation ExchangeAnion ExchangeHydrophobic InteractionMixed-modeHydroxyapatiteDye Affinity
Bulk Operations
FlocculationDetergent Viral Inactivation*Low pH Viral Inactivation*Heat InactivationPEGylationEnzymatic Reactions
Filtration Operations
Viral Filtration*Depth FiltrationTangential Flow UF/DFSingle-pass TFFMembrane Adsorbers
* = dedicated viral clearance
10/5/2017
F
RP
Viral Safety
Company Confidential © 2017 Eli Lilly and Company 8
Mission: To design/develop downstream purification processes with robust and consistent viral clearance capacity in support of clinical trials and commercialization
VirusFiltration
DetergentInactivation
Protein AChromatography
Low pHInactivation
IntermediateChromatography
= Dedicated viral clearance unit operations
• 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
PolishingChromatography
10/5/2017
Capture Chromatography - Protein A Affinity
Company Confidential © 2017 Eli Lilly and Company 9
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 withimproved selectivity and increased cleanability (base stability).
Protein AChromatography
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
10/5/2017
Intermediate Chromatography
Company Confidential © 2017 Eli Lilly and Company 10
Goal: To reduce and control multiple process and product related impurities.
IntermediateChromatography
Cation Exchange (typically bind/elute)• Manufacturing friendly – high load ratio, simple buffers• Strong HCP, Aggregate, DNA reduction, possible product-related impurities• Possible virus clearance
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
Polishing Chromatography
Company Confidential © 2017 Eli Lilly and Company 11
Goal: To provide an orthogonal mode of separation and serve as the final control point for multiple process and product related impurities.
PolishingChromatography
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)
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
Hydroxyapatite• Resin beads composed of crystalline Ca5(PO4)3(OH)• Strong Aggregate separation – but limited resin lifetime
10/5/2017
Case Study – Up-Front HCP Control
Company Confidential © 2017 Eli Lilly and Company 12
Problem: DNA and HCP reduction across Protein A are good, but can the affinity column perform even better?
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
Flocculation
10/5/2017
• 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
• Proprietary flocculation technique by Gagnon group at Bioprocess Institute, Singapore
• New stimulus reactive polymer from Merck Millipore (evaluated at Eli Lilly)
Case Study – 2-for-1 Inactivation
Company Confidential © 2017 Eli Lilly and Company 13
Problem: Some product molecules may be susceptible to enzymatic degradation by proteases that are expressed by mammalian cell culture
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
Heat Inactivation
10/5/2017
• 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 of enzymes takes advantage of differences in thermal stability between the product molecule and the enzymatic HCP
• May be performed in batch at lower temp/longer time – or continuously by HTST• Some viruses are also susceptible to heat treatment to achieve inactivation
Downstream Purification - Overview
Company Confidential © 2017 Eli Lilly and Company 14
Typical MonoclonalAntibody PurificationProcess
10/5/2017
F
RP
Production Bioreactor
ClarificationCentrifugation and Depth Filtration
0.2 micron
Viral Inactivation
Capture Chromatography
Low pH Viral Inactivation
Intermediate Chromatography
Polishing Chromatography
Virus Filtration
Tangential Flow UF/DF
DS Dispensing
DS Storage
Primary Recovery PurificationCell Culture
Purification