host cell protein clearance and detection: critical issues ......ich guidelines: regulatory...
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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! [email protected]