Development of Analytical Methodology for Intact Protein ...Development of Analytical Methodology for Intact Protein Separations: Understanding the Impact of Structure and Its Relation

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


– 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


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


– 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


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


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


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

Pfizer BioTherapeutics Pharmaceutical Sciences 8

“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


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


• 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


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


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)


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


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.


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


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


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


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


Intact Analysis - Nonporous Particle

MAb D

60C

MAb D MAb D

75C 90C


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


Reduced Analysis – Standard C8

MAb C

MAb D

60˚C

MAb C

MAb D

MAb C

MAb D

75˚C 90˚C


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


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


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


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


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


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)


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


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


Jason Starkey Ed Bouvier

Documents

Development of Analytical Methodology for Intact Protein ...Development of Analytical Methodology for Intact Protein Separations: Understanding the Impact of Structure and Its Relation