This document discusses the development and regulation of biological drugs from manufacturing to market approval. It covers the production of recombinant proteins through DNA transfection and cell culture. Extensive characterization and purification techniques are used to identify a drug's structure and activity. Biological drugs must undergo toxicity studies, clinical trials, and regulatory review and approval. Their immunogenicity and stability are important considerations throughout development and shelf life. Aggregates can induce unwanted immune responses if a drug denatures from manufacturing or storage issues. Maintaining proper formulation is key to preventing degradation and immunogenicity.

1. Biologicals
From development to market
Suzanne Vink-Hermeling
Qualified Person

2. Content
● Introduction
● Manufacturing
● Market Authorisation
● Immunogenicity
● Aggregates

3. Introduction - What are biologicals?
● Biologic = biological = biopharmaceutical = biologic
medical product
o any medicinal product manufactured in or extracted from biological
sources
● Composed of sugars, proteins, or nucleic acids
● Major groups
o Extracted from living tissue
o Produced by recombinant DNA techniques

4. 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

5. Manufacturing - Recombinant protein
● DNA
● Transfection
o eukaryotic cells
 animal
 plant
 human
o prokaryotic cells
● Cell culture
taken from http://www.bataviabioservices.com/production-services.php

6. Strain selection
Copied from “Handbook of Pharmaceutical Biotechnology, 2007”

7. Manufacturing - purification
● Disrupt cells
● Separate proteins from other cell parts
● Separate desired protein from other proteins

8. Purification techniques
● Filtration
● Precipitation
● Chromatography
o Size-exclusion
o Ion-exchange
o Reversed-phase
o Affinity

9. Manufacturing - Drug product
● Formulate
● Fill & Finish

10. Manufacturing - summary

11. Manufacturing - Scale up
● Lab-scale
o process development
o strain optimization
o medium optimization
● (pre)Clinical scale
o tox batches
o clinical phase I and II batches (need to be as good as tox batch)
● Commercial scale
o phase III / validation batches
o commercial batches

12. Bioreactors
lab-scale commercial scale

13. Market authorization
● Tox studies
● Clinical trials
● CMC
● Submission
● Approval
● Sales

14. Submission

15. Drug substance - Characterisation
● Structure determination
o primary structure
 amino acid sequence
o secondary structure
 α-helix; β-sheet
o tertiary structure
 how are side chains of aa structured
o quaternary structure
 how do monomers interact
● Active site
● Solubility

16. Structure determination
● Primary structure
o GPC
 Total mass (estimate)
o Mass-spectrometry
 Total mass
 Amino acid sequence
(using tryptic digests)
o Edman degradation
 N-terminal sequencing

17. Structure determination
● Secondary structure
o Circular dichroism
o FT-IR

18. Structure determination
● Tertiary structure
o Circular dichroism
o Fluorescence
o Mass spectrometry
● Quarternary structure
o GPC/SEC
o Native PAGE
Most of times only changes in structure can be determined

19. Drug Substance
● Activity
o Bioassay
● Content / Purity
o RP-HPLC
o GPC/SEC
o Electrophoresis
 capillary
 gel ((SDS)-PAGE)

20. Drug Substance - Stability
● Shelf life
● Chemical degradation
o Oxidation
o Deamidation
● Physical degradation
o Unfolding
o Aggregation
o Adsorption

21. Stability - shelf life
● ICH Q1
● Real-time
● Long-term
● Accelerated
● Analyses to include characterisation
● at end of shelf life prepare a DP batch

22. Chemical degradation
● Oxidation1
o cysteine, histidine, methionine, phenyalanine, tryptophan, tyrosine
o dissolved oxygen during manufacturing, traces of metal during
purification
o can induce structural changes
● Deamidation
o asparagine, glutamine
o pH>10 and high temperature
o can alter activity
1 Torosantucci et al; Pharm Res 2014 Mar 25;31(3):541-53

23. Physical degradation
● Unfolding
o Tertiary structure changes
● Adsorption
o concentration changes
● Aggregation
o quarternary structure changes

24. Submission

25. Analytical development
● Identity method
● Concentration method
● Impurity method
● Potency (if applicable)
● Pharmacopeial method
o pH
o Osmo
o Sterility
o Endotoxin (if applicable)
o Subvisible particles (if applicable)

26. Analytical method validation
● suitable for its intended use
● ICH Q2

27. Formulation development
● Stability
o prevent degradation
● Activity
o prevent immunogenicity
● Easy to use
o reduce number of injections
o route of administration

28. Formulation development
● Keep it simple!
● Understand the behaviour of your protein
o freeze/thaw
o temperature
o shear stress
● Will continue during clinical phase
➢ Specifications (ICH Q6)

29. Immunogenicity
The formation of antibodies against the administered
protein
● Binding antibodies
● Neutralizing antibodies

30. 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

31. Immune responses
Classical immune response Breaking of immune tolerance

32. Immunogenicity of rhIFNa2b
● rhIFNa2b was stressed
o oxidation (H2O2; metal-catalyzed)
o cross-linking with glutaraldehyde
o incubated in boiling water
● formulations were characterized
● formulations were injected (daily i.p. 3 weeks)
o wildtype mice
o transgenic mice immune tolerant for rhIFNa2b

33. Characterisation of formulations
Gel permeation chromatography

34. Characterisation - spectroscopy

35. Immunogenicity of rhIFNa2b
Titers of Ab recognizing native rhIFNa2b
wildtype mice transgenic mice

36. Immunogenicity of rhIFNa2b
no Abs present recognizing modified
rhIFNa2b in transgenic mice
Ab recognizing modified rhIFNa2b
wildtype mice

37. 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

38. Immunogenicity of rhIFNa2a
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

39. Aggregates - types
● Covalent aggregates
o Linked via chemical bonds
● Non-covalent aggregates
o Linked via non-chemical bonds
● Native aggregates
o protein monomers are in native state
● Non-native aggregates
o protein monomers are unfolded

40. Aggregates - mechanism
Taken from Monocloncal antibody aggregates; PhD thesis Vasco Filipe

41. Aggregates - analyses
● Light scattering techniques
● GPC
● MS
● (SDS)-PAGE
● Fluorescent dyes
Taken from Monocloncal
antibody aggregates; PhD
thesis Vasco Filipe

42. Aggregates - analyses
Taken from Monocloncal antibody
aggregates; PhD thesis Vasco
Filipe

43. Aggregate - Prevention
● Excipients
● Concentration
● Container

44. Summary
● Safety, efficacy and quality of protein therapeutics have
improved
● Product (drug substance) characterisation is important
● Formulation development is important
o to prevent degradation
o to prevent immunogenicity
● Aggregates can induce breaking of B-cell tolerance

45. Immunogenicity

46. Questions?
Suzanne Vink-Hermeling
http://www.svinx-consultancy.nl/
svink@svinx-consultancy.nl
+31 (0)6 48348112