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Designing an Effective Formulation for the
Manufacture of Recombinant Albumin
Technology Transferfor
BiopharmaceuticalsAmsterdam April 2006
Typical Excipients for Biopharmaceuticals
Trehalose Histidine
Mannose Aspartic acid
Sucrose Alanine
Sugars
Dextrose
Amino Acids
Glutamic acid
Sorbitol Polysorbate
Mannitol Albumin
Polyols
Glycerol
Polymers
Gelatin
Trials and Tribulations
Formulation The Clinic
Recombinant albumin – the background
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
Yeast – Positive Attributes
• GRAS status– S. cerevisiae– K. lactis
• Wide range of strains• Extensive industrial history
– 16 S. cerevisiaetherapeutic products marketed
– 7 P. pastoris therapeutic products under development
Gerngross, T. (2004) Nature Biotechnology 22, 1409-1414
8m3 working volume fermentation vessel
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
High Cell Density Fermentation System
• Constitutive expression1 2 3 4 5 61u
g
1ug
LaneFeed 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 supernatant12% Bis-Tris SDS Novex gel
MES Buffered
Purification of rHA
• Extracellular product• Multi-stage chromatography process• Simple step elution processes• All operations at room temperature
Recombumin® Purification
R&D Storage and Stability studies
R&D Final Container Stability Trials
• Monitored– Purity– Degradation– Dimer/oligomer– Free thiol– Thermal stability– Particle formation
Proteolysis and N-terminal Clipping-separate issues
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
Proteolysis and N-terminal Clipping-separate issues
YQAFAILVLAKFNEEGLDKFRHAS
KHAD
E VVCTKAFETVENVLKVHDEFPCQQ
L
AD
ENCDK
E S
SLHTLFGDKLCTVATL
ER
YT
GEMADCCAKQ
A
ERNECF
E P
L QHKDDNPNLPRLVRPEVDV M
CTAFHDNEETFLKKYLYEIAR
H P
FYAPELLFFAKRYKAAFTECCQAA D
KAACLLPK
A Q H D F A L K N
LDELR E
D
GKASS
KQRL
KCASL
KFGERAFKAWAVARLSQ K
AEFAEVSKLVTDLTKV
TECCHG
DLLECAD
RADLAKYICENQDSI
R PF
R Y
SSKLKECCEKP E
K
L L
SHCIAEV N
E
DEM
AP
DLPSLAAD
VESKD V
CKNY
EAKDVFLGMFLYEYARRH P
DYSVVLLLRLAKTYETT
EKCCAAA D
PHECYA
VFDEFK L
V
P
EEPQ
L
Domain 1 Domain 2 Domain 3
E
E G D F V E GL
IKQ C
E
N
LF
QLGEYKFQNALLVRYTK
PKQ
V
VSTPTLVEVSRNLGKV
SKCCKHPE A
KRMPCAE
YLSVVLNQLCVLHEKTP S
D
V
RVTKCCTESL V
NRRPCFSAL E
VDETYVPKEFNAET
FT
HAD I
CTLSEKERQIKKQTALV
LVKHK P
KATKEQLKAVMDDFAAF
EKCCKAD D
KETCFA
EGKKL
AVSQAAL
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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
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
Electrospray Mass Spectrometry
Recombumin®
HSA
Unmodified monomer
Monomer + blocked free thiol (cys34 + cys)
Monomer lacking N-terminal Asp-Ala
Monomer lacking C-terminal Leu
65500 66000 66500 67000 67500
DesAsp-Ala + blocked free thiol
Recombumin®
HSA
Unmodified monomer
Monomer + blocked free thiol (cys34 + cys)
Monomer lacking N-terminal Asp-Ala
Monomer lacking C-terminal Leu
65500 66000 66500 67000 67500
DesAsp-Ala + blocked free thiol
____
____
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
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
N-terminal Degradation of Albumin
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
Dimerisation and Oligomerisation
GP-HPLC
AB
SOR
BA
NC
E (2
80nm
)
0 2 4 6 8 0 12 TIME (minutes)
POLYMER
TRIMER
DIMER
MONOMER
RECOMBUMIN®
HSA
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)
Dimer Trimer and Cys34 Environment
Stewart et al Febs J (2005) 272 353-362
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
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
Free Thiol Stability Testing
Free Thiol Stability Data
0.15
0.25
0.35
0.45
0.55
0.65
0.75
0.85
0.95
0 5 10 15 20 25 30 35 40
Months
Free
Sh
mol
/mol
2-8oC
25+2oC
Development of a Heat stable Formulation and Prevention of Particle Formation
Recombumin® Formulation
• 20% (w/v) rHA• 130 - 160mM Sodium• 32mM Octanoate• 15mg.L-1 Polysorbate 80• Water for Injection
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
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
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
Heat Treatment of rHA Formulations
25% (w/v) rHA
Heated at 57ºC for 50hr
1. 40mM octanoate +15g.L-1
polysorbate 802. 40mM octanoate3. 20mM octanoate +
15g.L-1 polysorbate 804. 20mM octanoate
1 2 3 4
Formulation Stabilisation with Octanaote
HSA(mg.mL-1) (mM) (mmole.g-1 protein)
Armour Pharma 200 16 0.0845 7.2 0.16
200 32 0.16Novo Nordisk 50 20 0.4Protein Fractionation Centre
200 36 0.18
Manufacturer Octanoic acid
Blood Products Laboratory
DSC for Formulation Development
Octanoate concentration and Tmelt
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
Recombumin® Formulation
• 20% (w/v) rHA• 130 - 160mM Sodium• 32mM Octanoate• 15mg.L-1 Polysorbate 80• Water for Injection
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
Regulatory Approval
• Currently Recombumin® is used in– medical device coatings– IVF reagents– FDA approved manufacture of MMRII
vaccine– EMEA approved MMRII vaccine
