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/svink@svinx-consultancy.nl+31 (0)6 48348112