Host cell Protein Clearance and Detection: Critical issues for Biopharmaceutical development

Protein Quantification workshop, Waters: 13th November 2012

Vincent Monchois, R&D and Pilot Services Manager, Novasep

Corporate Presentation – 04/2012 2

A Global Presence

Seneffe and Gosselies - BELGIUM

Pompey - FRANCE

Le Mans - FRANCE

Chasse-sur-Rhône - FRANCE

Saint-Maurice-de-Beynost - FRANCE

Mourenx - FRANCE

Leverkusen - GERMANY

Shanghai - CHINA

Freeport THE BAHAMAS

Shrewsbury MA - USA

Boothwyn PA - USA

Chasse-sur-Rhône Mourenx

Freeport

S.-M.-de-Beynost

Shanghai

Pompey Seneffe

Le Mans

Shrewsbury Boothwyn

Leverkusen

Novasep Synthesis (Synthetic molecules) Novasep Process (Biomolecules)

Over 100 R&D projects per year Over 100 active molecules produced per year

Over 2,000 purification systems installed worldwide

Biopharma Functional Ingredients

Bio-Industries Food

Ingredients

Novasep Process: Mission and Markets

Your Process and/or Manufacturing

Needs

Technical Package

Novasep Process Development

R&D

Pilot Scale

Novasep Outsourcing Services

Novasep Equipment, Systems,

Consumables

Technologies and Competences: Focus on Biopharmaceutical applications

Therapeutic Biologics Products : Characteristics

Products made by or composed of viable organisms

− Natural & rDNA Proteins, mAb

− Vaccines, Cell and Gene Therapy

− Blood and Blood Derivatives

Majority of Biotech Products used genetically Living Cells

− Insert gene

− Specific conditions for optimal culture growth (Temp pH D02..)

− Complex Purification Steps

Difficult to demonstrate purity, identity, potency & consistency

Therapeutic Biologic Products vs small Molecule drugs

Complex structure and heterogeneous composition − Amino acid sequence

− Post translational modification (glycosylation, oxidation..)

− Conformation

− Stabilities issue (i.e. aggregation, proteolysis)

Complex production medium

− Medium components

− Cell components (i.e. DNA, lipids, proteins…)

− Complex Purification Steps

Potential entry gate for diseases

−Infectious diseases: Viral safety (i.e retrovirus, adventidious virus)

- Pyrogenic issues (i.e. endotoxin)

- Allergic issues (i.e. epitopes from HCP)

- Gene transfer (i.e. residual DNA)

taxol mAb

Drug Development Process

Periapproval

Phase III Phase II Research

Discovery Development

Phase I Phase IV

Preclinical

IND

Filed

Phase IIIb

NDA

Filed

NDA

Approved

Investigational New Drug (IND) - Insure identification, quality, and strength of the drug Impurities, sterility (CMC)

- Pharmacology/Toxicology Data

New Drug Application (NDA)

Increasing GMP requirements up to process and method validation

CMC: Chemistry, Manufacturing, and Controls

• Drug Substance: New Chemical Entity (NCE), Test Article, Active Pharmaceutical Ingredient (API): Material included the drug product, product-related substances, product- /-process-related impurities.

• Drug Product: Formulated Drug, including container and packaging

Drug substance characterization

Host cell protein = Process related impurity

Safety

Purity & Characterization - Well defined and controlled manufacturing process removing process-related

impurities

- Product-related impurities

- Product substances (product variants that are active)

Identity

Acceptance criteria for release and stability attributes should be established

ICH Guidelines: Regulatory requirement for HCP (i)

ICH Q7. Good Manufacturing Practice guide for active pharmaceutical ingredients, 2000

“18.17. Where appropriate, the removal of media components, host cell proteins, other process-related impurities, product-related impurities and contaminants should be demonstrated”

ICH Q11. Development and manufacture of drug substances (chemical entities and biotechnological/biological entities), 2012

3.1.4 Drug Substance Critical Quality Attributes Impurities are an important class of potential drug substance CQAs because of their potential impact on drug product safety. (…) For biotechnological/biological products, impurities may be process-related or product-related (see ICH Q6B), (…) . (e.g., Host Cell Proteins (HCP) …)

ICH Q6B. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, 1999

2.1.4 (…) Individual and/or collective acceptance criteria for impurities (product-related and process-related) should be set, as appropriate (…)

4.1.3 (…) Process-related impurities (…) may include cell culture media, host cell proteins, DNA, (…). These impurities should be minimized by the use of appropriate well-controlled manufacturing processes. (…)

6.2.1 (…) Cell substrate-derived impurities include, but are not limited to, proteins derived from the host organism (…). For host cell proteins, a sensitive assay e.g., immunoassay, capable of detecting a wide range of protein impurities is generally utilized.

ICH Guidelines: Regulatory requirement for HCP (ii)

ICH Q5E (quality of biotechnological product). Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process, 2004

2.2.1. Analytical techniques (…) It can be difficult to ensure that the chosen set of analytical procedures for the pre-change product will be able to detect modifications (…)

Consequently, the manufacturer should determine (…) whether or not existing tests remain appropriate (…)

For example, when the manufacturing process change gives rise to a different impurity profile in the host cell proteins, manufacturers should confirm that the test used to quantitate these impurities is still suitable for its intended purpose. It might be

appropriate to modify the existing test to detect the new impurities

ICH Guidelines: Regulatory requirement for HCP (iii)

PROCESS: Efficient and robust process maximizing HCP removal

Individual operations characterized

Target range for consistent removal

CONTROLS: Appropriate analytical procedures developed and validated

Sensitive

Able to detect potential of change in Manufacturing process assessed (comparability)

PRODUCT: Specifications set for the drug substance

Lower level as possible (ng/mg product)

ICH Guidelines: Regulatory requirement for HCP: Conclusions

USP: Expression system and safety

E. Coli Yeast CHO

VIRAL + - +++

ENDOTOXIN +++ - -

INTRACELLULAR (HCP) >> 900 mg/g Not used Not used

EXTRACELLULAR (HCP) No expression 200 mg/g 100 mg/g (100 000 ppm)

HCP= 0.01 mg/g (1 ppm) to 0.1 mg/g (100 ppm)

DSP: Overall Strategy of Biopharmaceutical product purification

DNA Endotoxine

Viral HCP

DNA Endotoxine

Viral HCP

DNA Endotoxine

Viral HCP

Current analytical methods used for HCP detection

BIOPHARM, Volume 13, Number 6, pages 38-45, May 2000

Standard method: Immunossay (ELISA)

HCP Quantification by ELISA: Development and Validation issue (i)

Typical source: Polyclonal antibody against cell lysate of CHO cells grown in serum supplemented media

Accuracy ?

Specificity ?

Precision?

Robustness?

Sensitivity?

Are anti-CHO HCP antibodies can detect HCP

in MY drug substance (?)

HCP Quantification by ELISA: Development and Validation issue (ii)

Immunization Polyclonal Antibodies

CHO Lysates

DETECTED

BLOCKED

NOT DETECTED

NOT DETECTED

Are anti-CHO HCP antibodies can detect HCP in MY drug substance sample (?)

Approaches to develop and validate HCP analytical methods

Test study to determine if all the HCP present in the drug substance are detected by anti-CHO prior phase III validation

Potential changes with production and purification procedures

Develop a Product specific Immunossay

Potential changes with production and purification procedures (new assay)

Time and cost consuming (12 months development)

Proteomics derived methods => LC/MS derived methods

Accurate and Specific: Bioinformatic support

Precise

Robust : not subjected to Process change

Sensitive: ppm range

Approaches to develop and validate HCP analytical methods

Approaches to develop and validate HCP analytical methods

Accuracy of anti-CHO HCP antibodies kit used since tox stage Alternative specific solutions to detect HCP for late stage/commercial stages

Trypsin digest followed by 2D LC/MSE approach: Digestion

Spiking with rabbit phosphorylase a (PHO) as internal standard

Trypsin digest followed by 2D LC/MSE approach: HPLC

Trypsin digest followed by 2D LC/MSE approach: MS analysis

Trypsin digest followed by 2D LC/MSE approach: Bioinformatic analysis

Trypsin digest followed by 2D LC/MSE approach: HCP in Crude

Trypsin digest followed by 2D LC/MSE approach: HCP in F2

Trypsin digest followed by 2D LC/MSE approach: HCP in concentrated drug

substance

Trypsin digest followed by 2D LC/MSE approach: Summary data

Conclusions:2D LC/MSE and HCP analytic development and validation for Biopharmaceutical

Detection and Identification of HCP up to 1 ppm

Proprietary and personal HCP data

Robust and Fast but may require further optimization

Data mining for Immunoassay or MRM development

Immunoassay: development of dedicated or validation of the broad kit

Liquid Chromatography coupled with tandem quadruple mass spectrometry (LC/MS/MS) in multiple reaction monitoring (MRM)

Gives insight for USP or DSP improvement

Thank you for your attention! vincent.monchois@novasep.com