6. Recombinant Human Albumin
Structure of rHA with five molecules of
myristate bound.
Curry et al. (1998) Nature Structural
Biology 5, 827-835
• Large secreted
protein
– 67kDa
– 585 amino
acids
• Highly folded
– 35 cysteines
– 17 disulphide
bonds
– 1 free cysteine
7. Yeast – Positive Attributes
• GRAS status
– S. cerevisiae
– K. lactis
• Wide range of strains
• Extensive industrial history
– 16 S. cerevisiae
therapeutic products
marketed
– 7 P. pastoris therapeutic
products under
development
Gerngross, T. (2004) Nature
Biotechnology 22, 1409-1414
8m3
working volume fermentation vessel
8. The Delta Expression Platform
• Expression vector development
– Native 2µm-based plasmid
– LEU2 selective marker
– Expression cassette
• Yeast strain development
– Highly developed family of
Saccharomyces cerevisiae strains
– Random and specific mutagenesis
9. High Cell Density Fermentation System
• Constitutive expression
1 2 3 4 5 6
1ug
1ug
Lane
Feed Time
(hr)
Feed Vol
(L)
Biomass
(g CDW/L)
1 6.5 0.1 8.9
2 14.0 0.3 14.9
3 30.5 1.1 46.8
4 38.3 1.9 67.5
5 54.5 4.8 101.8
6 55.5 5.0 101.3
Analysis of HCD culture supernatant
12% Bis-Tris SDS Novex gel
MES Buffered
10. Purification of rHA
• Extracellular product
• Multi-stage chromatography process
• Simple step elution processes
• All operations at room temperature
15. R&D Final Container Stability Trials
• Upon storage in the final container
an extra protein species was found
to be present
– time and temperature dependent
formation
– unaffected by pasteurisation or
protease inhibitors
16. Proteolysis and N-terminal Clipping
-separate issues
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17. Proteolysis in Culture Supernatant and
YAP3 Gene Deletion
1 2
rHA
monomer
45kDa
fragment
rHA produced in fed-batch
fermentations:
Lane 1: rHA produced
by YAP3 strain
15% fragment
Lane 2: rHA produced
by yap3 strain
1-5% fragment
18. Proteolysis and YAP3 Gene Deletion
• YAP3 deletion resulted in
– Increased rHA productivity in the
fermenter, more full length albumin
– Increased downstream recovery
• Less fragment improved recovery from a
step used to remove fragment by re-
optimising elution conditions
20. N-terminal Degradation of Albumin
• Loss of first two residues - Asp, Ala
– Temperature dependent
– Dependent on N-terminal α-amino group
– Metal independent
– Sequence (species) dependent
• Mechanism proposed
– Chan et al. (1995) Eur. J. Biochem. 227,
524-528
21. N-terminal Degradation of Albumin
• Proton withdrawal from α-amino group by
the Asp1 COOH
• Nucleophilic attack by α-amino nitrogen on
Ala2-His3 peptide carbonyl results in
cleavage of peptide bond and release of
cyclic peptide
23. N-terminal Degradation of Albumin
• Take home message
– N-terminal Degradation of Albumin is a
natural phenomenon exhibited by HSA
and recombinant albumin
– It cant be solved by optimisation of
formulation conditions
– Rate of formation can be reduced by
storage at 2-8oC
26. Polymer in HSA
• Polymer is formed in HSA by heat
treatment at 60oC for 10 hour –
pasteurisation
– Composed of heat denatured protein
contaminants as HSA need only be
>96% pure (USP)
27. Dimer Trimer and Cys34 Environment
Stewart et al Febs J (2005) 272 353-362
28. Dimer and Oligomer Formation
• Directed through Cys34
– Three types of dimer
• Non covalent, dissociated by SDS
• Covalent
– Reducible by mercaptoethanol
– Non reducible by mercaptoethanol
– Trimer and higher oligomers
• Formed through thiol disulphide interchange
• Oligomer formation is a natural phenomenon
and is time, temperature and concentration
dependent
29. Other Free Thiol Interactions
• Storage changes in free thiol
– Oxidation in the vial
• Oxygen in the headspace of the vial is finite
• Vial geometry and fill volume affect the
extent of oxidation
33. Protein Particle Formation
• Liquid formulation of proteins
– Denaturation at air liquid interface
• Agitation*
– Vessel agitation, on multiple cycle chromatography,
only agitate once at the end of the process step
• Foaming*
– Ensure dip pipes in vessels and return pipes in UF rigs
are configured properly
• Stress at the hydrophobic/hydrophilic interface in the
vial
– Not easily solved
*Especially with process scale equipment
34. Protein Particle Formation
• Particle formation in the final container
can be prevented by the addition of
non ionic surfactants, e.g. Polysorbate
80, Pluronate etc.
• Polysorbate 80 more effective at lower
concentrations
– Available animal free
35. Octanoate as a Stabiliser and Potential
Batch Tests – for therapeutic HSA
• 57oC / 50h, one bottle from a batch of
HSA
– Stability and purity
• 60oC / 10h, whole batch
– Pasteurisation, viral inactivation
• 30oC 2 weeks
– Sterility
39. Choice of Formulation Excipients
• USP
– 0.08mmol.g-1 protein N-acetyltryptophan
required for HSA therapeutic plus
equimolar octanaote
• DSC did not show any enhancement of
stability, therefore excluded
• Up to 0.4mmol.g-1 protein used in
octanaote only formulation
– DSC identified maximal heat stability at
0.16mmol.g-1 protein
41. Regulatory Approval
• Formulation Issues
– NTD, free thiol oxidation and dimer
formation are accepted as natural
phenomena, occurs in HSA and rHA
– Particle formation is prevented by
Polysorbate 80
– Heat stability optimised by use of DSC to
choose octanoate concentration
42. Regulatory Approval
• Currently Recombumin® is used in
– medical device coatings
– IVF reagents
– FDA approved manufacture of MMRII
vaccine
– EMEA approved MMRII vaccine
