process removal of impurities in biotech products · detergent, flocculant, leached protein a,...

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


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


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


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


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


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


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



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



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


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


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


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



Virus Filtration

Tangential Flow UF/DF

DS Dispensing

DS Storage

Primary Recovery PurificationCell Culture

Purification

process removal of impurities in biotech products · detergent, flocculant, leached protein a,...

Documents