Development of Analytical Methodology for Intact Protein Separations: Understanding the Impact of Structure and Its Relation to Performance – A Work in Progress Pfizer BioTherapeutics Pharmaceutical Sciences N.A. Lacher , Q. Wang, and C.W. Demarest June 24th, 2010
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Development of Analytical Methodology for Intact Protein Separations: Understanding the Impact of Structure and Its Relation to Performance – A Work in Progress
Pfizer BioTherapeutics Pharmaceutical Sciences
N.A. Lacher, Q. Wang, and C.W. Demarest
June 24th, 2010
Topics for Discussion
• Sample Complexity• Platform Analytical Methods for MAb Analysis• Platform RP Methods for MAb Analysis
– Statement of the problem – Disulfide Isomers
RP evaluation
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– RP evaluation
• Conclusions• Acknowledgements
Sample Complexity
Variable regionsLight chain
Heavy chain
CDR’sHypervariableregions
Antigen binding
Theoretical Molecular Mass ~150,000 Da>200 amino acid residues light chain>450 amino acid residues heavy chainMore than 1 predominant mass
Glycosylated:Complex Structure
Biantennary+/- Core Fucose
Sialylation
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Hinge region
Constant regions
Complimentactivation
Macrophagebinding
Carbohydrate side chain
y
Disulfide Bonds:Contain Inter and intra-chain bonds
1 Kyte, J. and R. Doolittle, J. Mol. Biol. 1982, 157, 105-132.
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Hydrophobicity Plot - Heavy Chain of an IgG MAb
Intact Analysis – Standard C8
MAb C
MAb D
MAb C
MAb D
MAb C
MAb D
60˚C 75˚C 90˚C
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Minutes2.00 4.00 6.00 8.00 10.00 12.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00
MAb A
MAb B
MAb A
MAb B
MAb A
MAb B
Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm)MPA: 0.1% TFA in H2OMPB: 0.085% TFA, 90% ACN in H2OFlow Rate: 0.2 mL/minGradient: 35-65%B in 15 minutes
Overall Hydrophobicity:MAb A (IgG2) = -296.9MAb B (IgG2) = -264.9MAb C (IgG1) = -283.8MAb D (IgG1) = -250.4
Intact Analysis – Superficially Porous
MAb C
MAb D
MAb C
MAb D
MAb C
MAb D
60˚C 75˚C 90˚C
Pfizer BioTherapeutics Pharmaceutical Sciences
Minutes2.00 4.00 6.00
Minutes2.00 4.00 6.00
Minutes2.00 4.00 6.00
MAb A
MAb B
MAb A
MAb B
MAb C
MAb A
MAb B
MAb C
Column: Agilent Zorbax Poroshell 300SB C8 (5 μm, 2.1 x 100 mm)MPA: 0.1% TFA in H2OMPB: 0.085% TFA, 90% ACN in H2OFlow Rate: 0.5 mL/minGradient: 35-65%B in 15 minutes
Overall Hydrophobicity:MAb A (IgG2) = -296.9MAb B (IgG2) = -264.9MAb C (IgG1) = -283.8MAb D (IgG1) = -250.4
Intact Analysis - Nonporous Particle
MAb D
60C
MAb D MAb D
75C 90C
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MAb A
MAb BMAb C
MAb A
MAb BMAb C
Minutes3.00 4.00 5.00 6.00 7.00 8.00
Minutes3.00 4.00 5.00 6.00 7.00 8.00
Minutes3.00 4.00 5.00 6.00 7.00 8.00
MAb A
MAb B
MAb C
Column: Imtakt Presto FF-C18 non-porous particle (2 μm, 2.1 x 150 mm)MPA: 0.1% TFA in H2OMPB: 0.085% TFA, 90% ACN in H2OFlow Rate: 0.2 mL/minGradient: 35-65%B in 15 minutes
Overall Hydrophobicity:MAb A (IgG2) = -296.9MAb B (IgG2) = -264.9MAb C (IgG1) = -283.8MAb D (IgG1) = -250.4
Reduced Analysis – Standard C8
MAb C
MAb D
60˚C
MAb C
MAb D
MAb C
MAb D
75˚C 90˚C
Pfizer BioTherapeutics Pharmaceutical Sciences
Minutes2.00 4.00 6.00 8.00 10.00 12.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00
MAb A
MAb B
MAb A
MAb B
MAb A
MAb B
Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm)MPA: 0.1% TFA in H2OMPB: 0.085% TFA, 90% ACN in H2OFlow Rate: 0.2 mL/minGradient: 35-65%B in 15 minutes
Note: LC and HC co-elute with this gradient for MAb D
Overall Hydrophobicity:MAb A (IgG2) LC = -104.7, HC = -192.2MAb B (IgG2) LC = -111.9, HC = -153.0MAb C (IgG1) LC = -87.4, HC = -196.8MAb D (IgG1) LC = -80.9, HC = -169.5
FabRICATOR Analysis – Standard C8
MAb C
MAb D
MAb C
MAb D
MAb C
MAb D
60˚C 75˚C 90˚C
Pfizer BioTherapeutics Pharmaceutical Sciences
Minutes2.00 4.00 6.00 8.00 10.00
Minutes2.00 4.00 6.00 8.00 10.00
Minutes2.00 4.00 6.00 8.00 10.00
MAb A
MAb B
MAb A
MAb B
MAb A
MAb B
Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm)MPA: 0.1% TFA in H2OMPB: 0.085% TFA, 90% ACN in H2OFlow Rate: 0.2 mL/minGradient: 35-65%B in 15 minutes
Overall Hydrophobicity:MAb A (IgG2) Fc = -129.5, F(ab')2 = -167.4MAb B (IgG2) Fc = -129.5, F(ab')2 = -135.4MAb C (IgG1) Fc = -130.7, F(ab')2 = -144.8MAb D (IgG1) Fc = -139.4, F(ab')2 = -119.7
Reduced FabRICATOR Analysis –Standard C8
MAb C
MAb D
MAb C
MAb D
MAb C
MAb D
60˚C 75˚C 90˚C
Pfizer BioTherapeutics Pharmaceutical Sciences
Minutes2.00 4.00 6.00 8.00 10.00
Minutes2.00 4.00 6.00 8.00 10.00
Minutes2.00 4.00 6.00 8.00 10.00
MAb A
MAb B
MAb A
MAb B
MAb A
MAb B
Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm)MPA: 0.1% TFA in H2OMPB: 0.085% TFA, 90% ACN in H2OFlow Rate: 0.2 mL/minGradient: 35-65%B in 15 minutes
Overall Hydrophobicity:MAb A (IgG2) LC = -104.7, FC = -129.5, Fd = -62.7MAb B (IgG2) LC = -111.9, Fc = -129.5, Fd = -23.5MAb C (IgG1) LC = -87.4, Fc = -130.7, Fd = -57.4MAb D (IgG1) LC = -80.9, Fc = -139.4, Fd = -38.8
T = Theoretical (hydrophobicity), E = Experimental, Red = poor/no elution at 60C
Conclusions
• Mechanism for RP column interaction is currently not well understood
• MAbs with similar sequence have drastically different performance
• Studies show that poor column performance is isolated in the Fd
• Elutropic strength of the mobile phase and alkyl chain
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p g p ylength can be optimized to improve recovery (surfactants currently being evaluated)
• Higher temperature improves kinetics allowing RP to be used as a platform technology with the drawback that the protein may degrade during the separation
• More appropriate modeling studies that focus on column/antibody interactions must be generated under RP-like conditions to determine if localized hydrophobic regions are responsible for poor elution
Acknowledgments
Pfizer:Sandeep KumarBilikallahalli MuralidharaRuss RobinsJ St k