technology transfer for biopharmaceuticals amsterdam 2006
TRANSCRIPT
![Page 2: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/2.jpg)
Designing an Effective Formulation for the
Manufacture of Recombinant Albumin
Technology Transferfor
BiopharmaceuticalsAmsterdam April 2006
![Page 3: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/3.jpg)
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
![Page 4: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/4.jpg)
Trials and Tribulations
Formulation The Clinic
![Page 5: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/5.jpg)
Recombinant albumin – the background
![Page 6: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/6.jpg)
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
![Page 7: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/7.jpg)
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
![Page 8: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/8.jpg)
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
![Page 9: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/9.jpg)
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
![Page 10: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/10.jpg)
Purification of rHA
• Extracellular product• Multi-stage chromatography process• Simple step elution processes• All operations at room temperature
![Page 11: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/11.jpg)
Recombumin® Purification
![Page 12: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/12.jpg)
R&D Storage and Stability studies
![Page 13: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/13.jpg)
R&D Final Container Stability Trials
• Monitored– Purity– Degradation– Dimer/oligomer– Free thiol– Thermal stability– Particle formation
![Page 14: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/14.jpg)
Proteolysis and N-terminal Clipping-separate issues
![Page 15: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/15.jpg)
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
![Page 16: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/16.jpg)
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
A
![Page 17: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/17.jpg)
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
![Page 18: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/18.jpg)
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
![Page 19: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/19.jpg)
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
____
____
![Page 20: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/20.jpg)
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
![Page 21: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/21.jpg)
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
![Page 22: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/22.jpg)
N-terminal Degradation of Albumin
![Page 23: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/23.jpg)
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
![Page 24: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/24.jpg)
Dimerisation and Oligomerisation
![Page 25: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/25.jpg)
GP-HPLC
AB
SOR
BA
NC
E (2
80nm
)
0 2 4 6 8 0 12 TIME (minutes)
POLYMER
TRIMER
DIMER
MONOMER
RECOMBUMIN®
HSA
![Page 26: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/26.jpg)
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)
![Page 27: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/27.jpg)
Dimer Trimer and Cys34 Environment
Stewart et al Febs J (2005) 272 353-362
![Page 28: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/28.jpg)
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
![Page 29: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/29.jpg)
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
![Page 30: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/30.jpg)
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
![Page 31: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/31.jpg)
Development of a Heat stable Formulation and Prevention of Particle Formation
![Page 32: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/32.jpg)
Recombumin® Formulation
• 20% (w/v) rHA• 130 - 160mM Sodium• 32mM Octanoate• 15mg.L-1 Polysorbate 80• Water for Injection
![Page 33: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/33.jpg)
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
![Page 34: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/34.jpg)
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
![Page 35: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/35.jpg)
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
![Page 36: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/36.jpg)
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
![Page 37: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/37.jpg)
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
![Page 38: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/38.jpg)
DSC for Formulation Development
Octanoate concentration and Tmelt
![Page 39: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/39.jpg)
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
![Page 40: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/40.jpg)
Recombumin® Formulation
• 20% (w/v) rHA• 130 - 160mM Sodium• 32mM Octanoate• 15mg.L-1 Polysorbate 80• Water for Injection
![Page 41: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/41.jpg)
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
![Page 42: Technology transfer for biopharmaceuticals amsterdam 2006](https://reader034.vdocument.in/reader034/viewer/2022052316/559c709d1a28ab59708b459f/html5/thumbnails/42.jpg)
Regulatory Approval
• Currently Recombumin® is used in– medical device coatings– IVF reagents– FDA approved manufacture of MMRII
vaccine– EMEA approved MMRII vaccine