N.A. Lacher, Q. Wang, and C.W. Demarest. June 24th, Pfizer BioTherapeutics Pharmaceutical Sciences

Transcription

1 Development of Analytical Methodology for Intact Protein Separations: Understanding the Impact of Structure and Its Relation to Performance A Work in Progress N.A. Lacher, Q. Wang, and C.W. Demarest June 24th, 2010

2 Topics for Discussion Sample Complexity Platform Analytical Methods for nalysis Platform RP Methods for nalysis Statement of the problem Disulfide Isomers RP evaluation Conclusions Acknowledgements

3 Sample Complexity Heavy chain Antigen binding Light chain CDR s Hypervariable regions Variable regions Theoretical Molecular Mass ~150,000 Da >200 amino acid residues light chain >450 amino acid residues heavy chain More than 1 predominant mass Glycosylated: Complex Structure Biantennary +/- Core Fucose Sialylation Hinge region Compliment activation Carbohydrate side chain Macrophage binding Constant regions Disulfide Bonds: Contain Inter and intra-chain bonds C-terminal Lysine Heterogeneity Additional Post-translations Modification (deamidation, methionine oxidation, etc.) Heterogeneity (9600) Heterogeneous in both size and charge

4 Platform Methods Platform Methods Available for: RP peptide mapping for PTMs/Identity CE-SDS (R and NR) SEC HCP DNA ph Appearance Concentration Carbohydrate analysis Recombinant Protein A ice Bioburden Endotoxin RP (Disulfide Isomers) Platform Methods Not Available for: Bioassay (specific to the target) RP intact/reduced method for PTMs, fragments, or identity

5 Rationale for a Platform RP Method RP Separation of omponents Fc Fc (R) Intact HC F(ab')2 LC Fd RP Shipping ID Method for MAbs MAb2 MAb3 MAb 4 MAb5 MAb6 MAb7 MAb1 MAb8 MAb9 0 MAb1 MAb11 MAb13 2 MAb Separation of clips (MS compatible) Separation of PTMs (MS compatible) Confirm retention time w/ ref. std. Simplicity compared to peptide mapping Small window for RP elution

6 IgG Performance by RP-LC IgG1 Acquity BEH300 C18 column (1.7 μm; 2.1 x 100 mm). AU Flow rate = 0.7 ml/min Gradient 25-40% B (4%/min) MPA 0.1% TFA in H 2 O MPB 0.1% TFA in ACN Column Temperature = 65 C IgG2 Acquity BEH300 C18 column (1.7 μm; 2.1 x 100 mm). AU Flow rate = 1.0 ml/min Gradient 19-60% B (8%/min) MPA 0.1% TFA in H 2 O MPB 0.1% TFA in ACN Column Temperature = 70 C Dillon, T. M., Bondarenko, P. V., Rehder, D. S., Pipes, G. D., et al., J. Chromatogr. A 2006, 1120,

7 IgG Molecules Fab 1 1 S S S S VL VH 138 C S S S S 198 CL CH1 218 S S Hinge { CHO CH2 224 s s S S s s 224 CH1 CL S S S S 218 CH2 VH VL S S S S S 22 S S S CHO 1 Fab Papain Cleavage Site His228/Thr229 1 IgG subclass IgG subclass properties 1 MW Amino acids in hinge Disulfide bonds in hinge IgG1 ~146 kda 15 2 IgG2 ~146 kda 12 4 IgG3 ~170 kda IgG4 ~146 kda 12 2 CH3 S S S S CH Fc 1 Salfeld, J. G., Nature Biotechnol. 2007, 25,

8 Disulfide Mediated Structural Isoforms Structures determined by proteolytic mapping and LC/MS 1 IgG2-A Classical Structure IgG2-B IgG2-A/B 1 Wypych, J., Li, M., Guo, A., Zhang, Z., et al., J. Biol. Chem. 2008, 283,

9 Optimized RP Separation for Disulfide Isomers B A/B Platform Method Agilent Poroshell 300SB C8 column (2.1 mm i.d. x 75 mm, 5 μm) A Gradient = 25% B to 34% B (1.5%/min) Column temperature = 85 C Flow rate = 1.5 ml/min Total run time = 10 minutes. Wang, Q., Lacher, N.A., Muralidhara, B.K., Schlittler, M.R., et al., J. Sep. Sci. 2010, In Press.

10 Chromatographic Development Particle characteristics particle size pore size porous, superficially porous, or nonporous van Deemter Eq. plate height mass transfer (c-term) 1 H = A + B u + Cu alkyl chain length (C4, C8, C18) Column length Temperature Pressure 2 Mobile phase composition (elutropic strength, ph) Denaturant 1 DeStefano, J.J., Langlois, T.J., Kirkland, J.J., J. Chromatogr. Sci., 2008, 46, Eschelbach, J.W., Jorgenson, J.W., Anal. Chem, 2006, 78,

11 Retention Relationships Weak chemical forces that govern protein conformation are also involved in chromatographic retention Not all amino acids in a protein can simultaneously contact the stationary phase Only residues at the surface can impact chromatographic behavior and only a fraction of the residues are involved with stationary phase interactions Heterogeneous distribution of residues on the surface allows some portions to dominate column behavior Structural changes that alter the protein surface can change behavior Interaction with the stationary phase can alter the protein secondary, tertiary, and quaternary structure Regnier, F.E., Science, 1987, 238,

12 Column Chemistry Vendor Morphology d p dimensions Pore Size (Å) Phase Surface Area m 2 /g 1 Agilent Superficially Porous 5 μm 2.1 x 75 mm 300 SB C Agilent Superficially Porous 5 μm 2.1 x 75 mm 300 ExtendC Agilent Porous 3.5 μm 2.1 x 100 mm 300 SB C Varian Porous 3 μm 2.0 x 150 mm 200 diphenyl Waters Porous 1.7 μm 2.1 x 150 mm 300 BEH C Imtakt Non-porous 2 μm 2.0 x 150 mm N/A C18 2

13 Sample Analysis Intact Reduced FabRICATOR FabRICATOR (Reduced) LC (2x) F(ab')2 Fd (2X) LC (2x) HC (2x) Fc (2x) Fc (2x) FabRICATOR cleaves hinge reagion after Gly 236

14 Influence of Temperature? Temperature 100 C 95 C 90 C 85 C Temperature 100 C 95 C 90 C 85 C 80 C 75 C 70 C 65 C 60 C 80 C 75 C 70 C 65 C 60 C 55 C 50 C 45 C 40 C 55 C 50 C 45 C 40 C 35 C 35 C 30 C 30 C Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm) MPA: 0.1% TFA in H 2 O MPB: 0.085% TFA, 90% ACN in H 2 O Flow Rate: 0.2 ml/min Gradient: 35-65%B in 15 minutes

15 Pressure Influence? Flow Rate (ml/min) Pressure v. Flow Rate y = 16424x R 2 = Pressure (psi) Flow Rate (ml/min) USP Plate Count Flow Rate (ml/min) Plate Count v. Flow Rate Flow Rate (ml/min) Waters Acquity UPLC BEH300 C4 (1.7 μm, 2.1 x150mm) Temperature = 60 C

16 Antibody Hydrophobicity? Kyte and Doolittle Hydrophobicity Values 1 PHE = 2.8 CYS = 2.5 SER = -0.8 ASN = -3.5 MET = 1.9 TRP = -0.9 PRO = -1.6 GLU = Kyte, J. and R. Doolittle, J. Mol. Biol. 1982, 157, ILE = 4.5 ALA = 1.8 TYR = -1.3 LYS = -3.9 LEU = 3.8 THR = -0.7 HIS = -3.2 ASP = -3.5 VAL = 4.2 GLY = -0.4 GLN = -3.5 ARG = -4.5 Hydrophobicity Plot - Heavy Chain of an IgG MAb

17 Intact Analysis Standard C8 60 C 75 C 90 C Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm) MPA: 0.1% TFA in H 2 O MPB: 0.085% TFA, 90% ACN in H 2 O Flow Rate: 0.2 ml/min Gradient: 35-65%B in 15 minutes Overall Hydrophobicity: (IgG2) = (IgG2) = (IgG1) = (IgG1) =

18 Intact Analysis Superficially Porous 60 C 75 C 90 C Column: Agilent Zorbax Poroshell 300SB C8 (5 μm, 2.1 x 100 mm) MPA: 0.1% TFA in H 2 O MPB: 0.085% TFA, 90% ACN in H 2 O Flow Rate: 0.5 ml/min Gradient: 35-65%B in 15 minutes Overall Hydrophobicity: (IgG2) = (IgG2) = (IgG1) = (IgG1) =

19 Intact Analysis - Nonporous Particle 60C 75C 90C Column: Imtakt Presto FF-C18 non-porous particle (2 μm, 2.1 x 150 mm) MPA: 0.1% TFA in H 2 O MPB: 0.085% TFA, 90% ACN in H 2 O Flow Rate: 0.2 ml/min Gradient: 35-65%B in 15 minutes Overall Hydrophobicity: (IgG2) = (IgG2) = (IgG1) = (IgG1) =

20 Reduced Analysis Standard C8 60 C 75 C 90 C Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm) MPA: 0.1% TFA in H 2 O MPB: 0.085% TFA, 90% ACN in H 2 O Flow Rate: 0.2 ml/min Gradient: 35-65%B in 15 minutes Overall Hydrophobicity: (IgG2) LC = , HC = (IgG2) LC = , HC = (IgG1) LC = -87.4, HC = (IgG1) LC = -80.9, HC = Note: LC and HC co-elute with this gradient for

21 FabRICATOR Analysis Standard C8 60 C 75 C 90 C Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm) MPA: 0.1% TFA in H 2 O MPB: 0.085% TFA, 90% ACN in H 2 O Flow Rate: 0.2 ml/min Gradient: 35-65%B in 15 minutes Overall Hydrophobicity: (IgG2) Fc = , F(ab')2 = (IgG2) Fc = , F(ab')2 = (IgG1) Fc = , F(ab')2 = (IgG1) Fc = , F(ab')2 =

22 Reduced FabRICATOR Analysis Standard C8 60 C 75 C 90 C Column: Agilent Zorbax 300SB C8 (3.5 μm, 2.1 x 100 mm) MPA: 0.1% TFA in H 2 O MPB: 0.085% TFA, 90% ACN in H 2 O Flow Rate: 0.2 ml/min Gradient: 35-65%B in 15 minutes Overall Hydrophobicity: (IgG2) LC = , F C = , Fd = (IgG2) LC = , Fc = , Fd = (IgG1) LC = -87.4, Fc = , Fd = (IgG1) LC = -80.9, Fc = , Fd = -38.8

23 Elution Order (Data at 75 C) Intact (T) Peak 1 MAb1 (-297) Peak 2 MAb2 (-294) Peak 3 MAb3 (-285) Peak 4 MAb4 (-284) Peak 5 MAb5 (-274) Peak 6 MAb6 (-272) Peak 7 MAb7 (-270) Peak 8 MAb8 (-265) Peak 9 MAb9 (-264) Peak 10 MAb10 (-255) Peak 11 MAb11 (-250) Intact (E) MAb2 MAb1 MAb3 MAb5 MAb7 MAb4 MAb11 MAb12 MAb9 MAb6 MAb10 MAb8 Peak 12 MAb12 (-237) LC (T) MAb8 (-112) MAb5 (-112) MAb3 (-107) MAb6 (-106) MAb1 (-105) MAb10 (-100) MAb2 (-99) MAb7 (-95) MAb12 (-91) MAb4 (-87) MAb11 (-81) LC (E) MAb5 MAb8 MAb6 MAb12 MAb2 MAb1 MAb4 MAb10 MAb7 MAb3 MAb11 MAb9 MAb9 (-76) HC (T) MAb4 (-197) MAb2 (-195) MAb1 (-192) MAb9 (-187) MAb3 (-178) MAb7 (-175) MAb11 (-170) MAb6 (-166) MAb5 (-162) MAb10 (-154) MAb8 (-153) MAb12 (-146) HC (E) MAb2 MAb1 MAb4 MAb11 MAb7 MAb9 MAb3 MAb5 MAb12 MAb6 MAb8 MAb10 F(ab')2 (T) F(ab )2 (E) MAb1 (-167) MAb2 (-163) MAb3 (-156) MAb6 (-150) MAb5 (-147) MAb4 (-145) MAb7 (-143) MAb12 (-143) MAb8 (-135) MAb10 (-124) MAb9 (-123) MAb11 (-120) MAb1 MAb2 MAb3 MAb4 MAb5 MAb7 MAb11 MAb12 MAb9 MAb6 MAb10 MAb8 T = Theoretical (hydrophobicity), E = Experimental, Red = poor/no elution at 60C

24 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 length 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

25 Acknowledgments Pfizer: Sandeep Kumar Bilikallahalli Muralidhara Russ Robins Jason Starkey Agilent Technologies: Sue D Antonio John Palmer Waters: Ed Bouvier