Biologicals (from development to market)
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Biologicals From development to market Suzanne Vink-Hermeling Qualified Person
description
An overview of the development and marketing of biologicals.
Transcript of Biologicals (from development to market)
BiologicalsFrom development to market
Suzanne Vink-HermelingQualified Person
Content
● Introduction● Manufacturing● Market Authorisation● Immunogenicity● Aggregates
Introduction - What are biologicals?
● Biologic = biological = biopharmaceutical = biologic medical producto any medicinal product manufactured in or extracted from biological
sources
● Composed of sugars, proteins, or nucleic acids● Major groups
o Extracted from living tissueo Produced by recombinant DNA techniques
Introduction - History
● Therapeutic proteins (1920 Insulin)o extracted from animal tissue
non-endogenous impure sample
o extracted from human tissue impurities
o recombinant proteins highly purified
This talk will be about therapeutic recombinant proteins
Manufacturing - Recombinant protein
● DNA
● Transfectiono eukaryotic cells
animal plant human
o prokaryotic cells
● Cell culturetaken from http://www.bataviabioservices.com/production-services.php
Strain selection
Copied from “Handbook of Pharmaceutical Biotechnology, 2007”
Manufacturing - purification
● Disrupt cells
● Separate proteins from other cell parts
● Separate desired protein from other proteins
Purification techniques
● Filtration
● Precipitation
● Chromatographyo Size-exclusiono Ion-exchangeo Reversed-phaseo Affinity
Manufacturing - Drug product
● Formulate
● Fill & Finish
Manufacturing - summary
Manufacturing - Scale up
● Lab-scaleo process developmento strain optimizationo medium optimization
● (pre)Clinical scaleo tox batcheso clinical phase I and II batches (need to be as good as tox batch)
● Commercial scaleo phase III / validation batcheso commercial batches
Bioreactors
lab-scale commercial scale
Market authorization
● Tox studies
● Clinical trials
● CMC
● Submission
● Approval
● Sales
Submission
Drug substance - Characterisation
● Structure determinationo primary structure
amino acid sequenceo secondary structure
α-helix; β-sheeto tertiary structure
how are side chains of aa structuredo quaternary structure
how do monomers interact
● Active site● Solubility
Structure determination
● Primary structureo GPC
Total mass (estimate)o Mass-spectrometry
Total mass Amino acid sequence
(using tryptic digests)o Edman degradation
N-terminal sequencing
Structure determination
● Secondary structureo Circular dichroism
o FT-IR
Structure determination
● Tertiary structureo Circular dichroismo Fluorescenceo Mass spectrometry
● Quarternary structureo GPC/SECo Native PAGE
Most of times only changes in structure can be determined
Drug Substance
● Activityo Bioassay
● Content / Purityo RP-HPLCo GPC/SECo Electrophoresis
capillary gel ((SDS)-PAGE)
Drug Substance - Stability
● Shelf life● Chemical degradation
o Oxidationo Deamidation
● Physical degradationo Unfoldingo Aggregationo Adsorption
Stability - shelf life
● ICH Q1● Real-time● Long-term● Accelerated● Analyses to include characterisation
● at end of shelf life prepare a DP batch
Chemical degradation
● Oxidation1
o cysteine, histidine, methionine, phenyalanine, tryptophan, tyrosineo dissolved oxygen during manufacturing, traces of metal during
purificationo can induce structural changes
● Deamidationo asparagine, glutamineo pH>10 and high temperatureo can alter activity
1 Torosantucci et al; Pharm Res 2014 Mar 25;31(3):541-53
Physical degradation
● Unfoldingo Tertiary structure changes
● Adsorptiono concentration changes
● Aggregationo quarternary structure changes
Submission
Analytical development
● Identity method● Concentration method● Impurity method● Potency (if applicable)● Pharmacopeial method
o pHo Osmoo Sterilityo Endotoxin (if applicable)o Subvisible particles (if applicable)
Analytical method validation
● suitable for its intended use● ICH Q2
Formulation development
● Stability o prevent degradation
● Activityo prevent immunogenicity
● Easy to useo reduce number of injectionso route of administration
Formulation development
● Keep it simple!● Understand the behaviour of your protein
o freeze/thawo temperatureo shear stress
● Will continue during clinical phase
➢ Specifications (ICH Q6)
Immunogenicity
The formation of antibodies against the administered protein● Binding antibodies● Neutralizing antibodies
Types of immune reactions
Reaction to neo-antigens (classical immune response)
Breakdown of immune tolerance
Properties of product Microbial or plant origin Human homologue
Cause of immunogenicity
Presence of non-self antigens
impurities and presence of aggregates; formulation
Predictive models Conventional animals transgenic immune tolerant mice
Consequences Loss of efficacy in majority of patients no consequences
Immune responses
Classical immune response Breaking of immune tolerance
Immunogenicity of rhIFNa2b
● rhIFNa2b was stressedo oxidation (H2O2; metal-catalyzed)o cross-linking with glutaraldehydeo incubated in boiling water
● formulations were characterized● formulations were injected (daily i.p. 3 weeks)
o wildtype miceo transgenic mice immune tolerant for rhIFNa2b
Characterisation of formulations
Gel permeation chromatography
Characterisation - spectroscopy
Immunogenicity of rhIFNa2b
wildtype mice transgenic mice
Titers of Ab recognizing native rhIFNa2b
Immunogenicity of rhIFNa2b
no Abs present recognizing modified rhIFNa2b in transgenic mice
Ab recognizing modified rhIFNa2b
wildtype mice
Conclusion
● Large aggregates of denatured protein are not necessarily more immunogenic than smaller aggregates composed of more native-like protein
● Both structure as well as size influence immunogenicity
A: lyophilized powder stored at RT
B: lyophilized powder stored at 4ºC
C: HSA-containing liquid stored at 4ºC
D: ultrapure HSA-free liquid formulation stored at 4ºC
E: ultrapure lyophilized powder
stored at 4ºC
Immunogenicity of rhIFNa2a
Aggregates - types
● Covalent aggregateso Linked via chemical bonds
● Non-covalent aggregateso Linked via non-chemical bonds
● Native aggregateso protein monomers are in native state
● Non-native aggregateso protein monomers are unfolded
Aggregates - mechanism
Taken from Monocloncal antibody aggregates; PhD thesis Vasco Filipe
Aggregates - analyses
● Light scattering techniques● GPC● MS● (SDS)-PAGE● Fluorescent dyes
Taken from Monocloncal antibody aggregates; PhD thesis Vasco Filipe
Aggregates - analyses
Taken from Monocloncal antibody aggregates; PhD thesis Vasco Filipe
Aggregate - Prevention
● Excipients
● Concentration
● Container
Summary
● Safety, efficacy and quality of protein therapeutics have improved
● Product (drug substance) characterisation is important● Formulation development is important
o to prevent degradationo to prevent immunogenicity
● Aggregates can induce breaking of B-cell tolerance
Immunogenicity
Questions?
Suzanne Vink-Hermeling
http://www.svinx-consultancy.nl/[email protected]+31 (0)6 48348112
