development of analytical methodology for intact protein ...development of analytical methodology...
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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
C-terminal Lysine HeterogeneityAdditional Post-translations Modification(deamidation, methionine oxidation, etc.)
Heterogeneous in both size and charge
Heterogeneity (9600)2 ≈ 108
http://www.fda.gov/ohrms/dockets/AC/05/slides/2005-4187S2_05_Cherney.ppt
Platform Methods
Platform Methods Available for:– RP peptide mapping for
PTMs/Identity– CE-SDS (R and NR)– SEC– HCP
DNA
– Concentration– Carbohydrate analysis– Recombinant Protein A– iCE– Bioburden
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– DNA– pH– Appearance
– Endotoxin– RP (Disulfide Isomers)
Platform Methods Not Available for:– Bioassay (specific to the target)– RP intact/reduced method for PTMs,
fragments, or identity
Rationale for a Platform RP Method
Fc (R)
Intact
LC
HCF(ab')2
Fd
RP Separation of MAb Components
MAb
8
MAb
1 MAb
13
MAb
2
2
MAb
4M
Ab3 MAb
5M
Ab6
MAb
90
MAb
7
MAb
11
RP Shipping ID Method for MAbs
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Minutes2.00 3.00 4.00 5.00 6.00
Fc
• Separation of clips (MS compatible)• Separation of PTMs (MS compatible)
Minutes19.00 20.00 21.00 22.00 23.00 24.00 25.00
MAb
1
MAb
1
• Confirm retention time w/ ref. std.• Simplicity compared to peptide mapping• Small window for RP elution
IgG Performance by RP-LCAU
- 0 .0 4
- 0 .0 2
0 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
0 .1 0
0 .1 2
0 .1 4
0 .1 6
0 .1 8
0 .2 0
0 .2 2
0 .2 4
0 .2 6
0 .2 8
0 .3 0
0 .3 2
0 .3 4
0 .3 6
0 .3 8
0 .4 0
0 .4 2
0 .4 4
0 .4 6
0 .4 8
0 .0 0 0 .5 0 1 .0 0 1 .5 0 2 .0 0 2 .5 0 3 .0 0 3 .5 0 4 .0 0 4 .5 0 5 .0 0 5 .5 0 6 .0 0 6 .5 0 7 .0 0 7 .5 0 8 .0 0 8 .5 0 9 .0 0 9 .5 0 1 0. 00
IgG1 Acquity BEH300 C18 column (1.7 μm; 2.1 x 100 mm).
Flow rate = 0.7 mL/minGradient – 25-40% B (4%/min)MPA – 0.1% TFA in H2OMPB – 0.1% TFA in ACNColumn Temperature = 65°C
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6
M in u te s0 0 0 0 5 0 0 0 5 0 0 0 5 0 3 0 0 3 5 0 0 0 5 0 5 0 0 5 5 0 6 0 0 6 5 0 0 0 5 0 8 0 0 8 5 0 9 0 0 9 5 0 0 00
AU
-0.08
-0.06
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
IgG2 Acquity BEH300 C18 column (1.7 μm; 2.1 x 100 mm).
Flow rate = 1.0 mL/minGradient – 19-60% B (8%/min)MPA – 0.1% TFA in H2OMPB – 0.1% TFA in ACNColumn Temperature = 70°C
Dillon, T. M., Bondarenko, P. V., Rehder, D. S., Pipes, G. D., et al., J. Chromatogr. A 2006, 1120, 112-120
IgG Molecules
IgG subclass
MW Amino acids in hinge
Disulfide bonds in
hingeIgG1 ~146 kDa 15 2
IgG subclass properties 1
Fab
SS
S
SS
SS S
S
SS
S
SS
SS
SS
SS
Fab
VL
VH
CL
CH1
VH
VLCH1
CL
1
1
1
1
23
92
22
96
138
148
204198
204
198
148
138
96
92
2223
224
224
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IgG1 146 kDa 15 2IgG2 ~146 kDa 12 4IgG3 ~170 kDa 62 11IgG4 ~146 kDa 12 2
1 Salfeld, J. G., Nature Biotechnol. 2007, 25, 1369-1372.
SS
SS
SS
SS
SS
S
s ss s
Fc
C
CH2CH2
CH3 CH3
Papain Cleavage Site His228/Thr229
{Hinge
CHO CHO
218
450 450
218
230233
Disulfide–Mediated Structural Isoforms
Structures determined by proteolytic mapping and LC/MS 1
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“IgG2-A”
Classical Structure
“IgG2-A/B”“IgG2-B”
1 Wypych, J., Li, M., Guo, A., Zhang, Z., et al., J. Biol. Chem. 2008, 283, 16194-16205.
Optimized RP Separation for Disulfide Isomers
Platform Method –Agilent Poroshell 300SB C8 column (2.1 mm i.d. x 75 mm, 5 μm)
Gradient = 25% B to 34% B (1 5%/min)
B
A/B
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Gradient = 25% B to 34% B (1.5%/min)Column temperature = 85 °CFlow rate = 1.5 mL/minTotal run time = 10 minutes.
Wang, Q., Lacher, N.A., Muralidhara, B.K., Schlittler, M.R., et al., J. Sep. Sci. 2010, In Press.
A
Chromatographic Development
Particle characteristics
• particle size
• pore size
• porous, superficially porous, or nonporous
• mass transfer (c-term)1CuuBAH ++=
van Deemter Eq. – plate height
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• alkyl chain length (C4, C8, C18)
Column length
Temperature
Pressure 2
Mobile phase composition (elutropic strength, pH)
Denaturant1 DeStefano, J.J., Langlois, T.J., Kirkland, J.J., J. Chromatogr. Sci., 2008, 46, 254-2602 Eschelbach, J.W., Jorgenson, J.W., Anal. Chem, 2006, 78, 1697-1706
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
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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, 319-323
Column Chemistry
Vendor Morphology dp dimensions Pore Size (Å) Phase Surface Area m2/g
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p gy p ( ) g
1 Agilent Superficially Porous 5 μm 2.1 x 75 mm 300 SB C8 4.5
2 Agilent Superficially Porous 5 μm 2.1 x 75 mm 300 ExtendC18 4.5
3 Agilent Porous 3.5 μm 2.1 x 100 mm 300 SB C8 45
4 Varian Porous 3 μm 2.0 x 150 mm 200 diphenyl 200
5 Waters Porous 1.7 μm 2.1 x 150 mm 300 BEH C4 88
6 Imtakt Non-porous 2 μm 2.0 x 150 mm N/A C18 2
Sample Analysis
Intact Reduced FabRICATOR® FabRICATOR® (Reduced)
Fd (2X) LC (2x)
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F(ab')2
Fc (2x)HC (2x)
LC (2x)
LC (2x)
Fc (2x)
FabRICATOR® cleaves hinge reagion after Gly236
Influence of Temperature?
MAb A MAb BTemperature
65˚C70˚C75˚C80˚C
85˚C90˚C95˚C
100˚CTemperature
65˚C70˚C75˚C80˚C
85˚C90˚C95˚C
100˚C
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Minutes14.00 16.00 18.00 20.00 22.00 24.00
Minutes14.00 16.00 18.00 20.00 22.00 24.00
30˚C
35˚C
40˚C45˚C50˚C55˚C
60˚C65˚C
30˚C
35˚C
40˚C45˚C50˚C55˚C60˚C
65 C
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
Pressure Influence?MAb A
y = 16424x + 450.29R2 = 0.9996
0
2000
4000
6000
8000
10000
12000
14000
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Pres
sure
(psi
)
Pressure v. Flow Rate
0.2
0.3
0.4
0.5
0.6
0.7
0.1
Flow Rate (mL/min)
Pfizer BioTherapeutics Pharmaceutical SciencesMinutes
5.00 10.00 15.00 20.00
Minutes6.00 8.00 10.00 12.00
MAb B
Flow Rate (mL/min)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Flow Rate (mL/min)
USP
Pla
te C
ount
Plate Count MAb A v. Flow Rate
0.2
0.3
0.4
0.5
0.6
0.7
0.1
Flow Rate (mL/min)
Waters Acquity UPLC® BEH300 C4 (1.7 μm, 2.1 x150mm)Temperature = 60˚C
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 = -3.5
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
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
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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
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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
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
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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
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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
Elution Order (Data at 75˚C)
Peak 1
Peak 2
Peak 3
Peak 4
Peak 5
Peak 6
Peak 7
Peak 8
Peak 9
Peak 10
Peak 11
Peak 12
Intact (T) MAb1(-297)
MAb2(-294)
MAb3(-285)
MAb4(-284)
MAb5(-274)
MAb6(-272)
MAb7(-270)
MAb8(-265)
MAb9(-264)
MAb10(-255)
MAb11(-250)
MAb12(-237)
Intact (E) MAb2 MAb1 MAb3 MAb5 MAb7 MAb4 MAb11 MAb12 MAb9 MAb6 MAb10 MAb8
LC (T) MAb8(-112)
MAb5(-112)
MAb3(-107)
MAb6(-106)
MAb1(-105)
MAb10(-100)
MAb2(-99)
MAb7(-95)
MAb12(-91)
MAb4(-87)
MAb11(-81)
MAb9(-76)
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LC (E) MAb5 MAb8 MAb6 MAb12 MAb2 MAb1 MAb4 MAb10 MAb7 MAb3 MAb11 MAb9
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)
MAb1(-167)
MAb2(-163)
MAb3(-156)
MAb6(-150)
MAb5(-147)
MAb4(-145)
MAb7(-143)
MAb12(-143)
MAb8(-135)
MAb10(-124)
MAb9(-123)
MAb11(-120)
F(ab’)2(E)
MAb1 MAb2 MAb3 MAb4 MAb5 MAb7 MAb11 MAb12 MAb9 MAb6 MAb10 MAb8
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
Agilent Technologies:Sue D’AntonioJohn Palmer
Waters:Ed Bouvier
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Jason Starkey Ed Bouvier