The Present State and Future Outlook for Characterizing the Higher Order Structure (HOS) of

Protein Drugs in the Biopharmaceutical Industry

Steven Berkowitz, Senior Principal Scientist

Biogen Idec

2nd International Symposium on the Higher Order Structure (HOS) of Protein Therapeutics

Feb. 11, 2013

Assessing the HOS in the Biopharmaceutical IndustryIndustry

HOS = secondary, tertiary & quaternary structure

Structure = spatial (3-D) & temporal (dynamics/flexibility)

2Lecture slide from Dr, Randall MauldinDepartment of Biochemistry and Biophysics, UNC Medicine

Points to Consider in Discussing the HOS of Protein Biopharmaceutical

1. Protein biopharmaceuticals are made using a living system (cell)(cell)

2. Chemically heterogeneous due to post-translational modification (PTMs) both in vivo & in vitro (degradation)modification (PTMs), both in vivo & in vitro (degradation)

3. Production lots of a biopharmaceutical "Cannot be made so they are identical”

4 Rather they are made “Highly Similar” in terms of key4. Rather they are made Highly Similar in terms of key attributes: structure, purity, efficacy and safety

5 M t f th HOS f t i i h ld t th b d5. Most of the HOS of a protein is held together by secondary (weak) bonds

3

In the Biopharmaceutical Industry What Do We Want Know in Characterizing the HOS of Protein Drugs

(Research, Process Development & Manufacturing)

1 Impact of primary structure changes on the HOS of the protein drug1. Impact of primary structure changes on the HOS of the protein drug (variants forms of the protein drug)

2. Detection & impact of silent changes on the HOS of protein drugp g p g

3. An understanding of the physico-chemical properties (stability) of aprotein drug by stressing it and studying changes to its HOS –formulation

4. Consistency of manufacturing (lot-to-lot), via consistency of the t i d ’ HOS (I t l) C bilit t diprotein drug’s HOS – (Internal) Comparability studies

5. Impact of process/site changes on the HOS of the protein drug –(internal) Comparability studies(internal) Comparability studies

6. Biosimilar HOS vs Innovator HOS – (External) Comparability studies4

Ideal Attributes of a Biophysical Tool/Method for Assessing the HOS in the Biopharmaceutical Industry

1. High precision & accuracy

2. Large response per unit change (high sensitivity)

3. Probes all the parts of the protein drug moleculep p g

4. High resolution information (detect small change in a molecule)

5. Minimal sample processing/handling – minimal matrix effects

6. Robustness, easy to use and level of expertise/training required not high

7. High sample throughput (measurement completed in a short time and automated measurement & analysis)

8. Multiple vendors, not expensive

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Basic Biophysical Toolbox Used Presently to Assess the HOS of Protein Drugs in the Biopharmaceutical Industry

Spectroscopic:UV-Vis – aromatic residuesFluorescence – aromatic residuesFluorescence aromatic residuesCD – amide bond (polypeptide backbone) & aromatic residuesFTIR – amide bond (polypeptide backbone)

Hydrodynamic (size):Hydrodynamic (size):SEC – aggregationAUC – aggregation, Sm, concentration-dependent aggregationLS (SLS & DLS) – aggregation

Thermodynamic:DSC – domain Tm, thermogram

Native Chromatography & electrophoresisIEC, HIC – surface properties IEF – charge

Binding:Via Biacore SPR, ITC, Fluorescence – drug target, co-factor (metal, etc.), dyes Biological – cell assay

6

Understanding the Uncertainty of Biophysical Measurements in Order to Assess Comparability

Far-UV CD

Understanding the Uncertainty of Biophysical Measurements in Order to Assess Comparability

DSC

8

Present Routine Spectroscopic Tools Used to Assess Biophysical Comparabilityp y p y

UV‐VIS                               Fluorescence1.2

RECD 16283-10-030

0.400.015

0.02

RS037-001RECD 17418-10-034

0.4

0.6

0.8

1

OD

nm-O

D38

0)/(O

D28

0-O

D38

0)

RECD 16283-10-037

RS028-001

0.20

0.30

resc

ence

Intens

ity (V

nm)

RECD 16283-10-030

RECD 16283-10-037

RS028-001

RECD 16283-10-030

RECD 16283-10-037

RS028-001-0.02

-0.015

-0.01

-0.005

0

0.005

0.01

240 260 280 300 320 340

Wavelength, (nm)

d2 (OD

nm)/d

(nm

)2

0

0.2

240 250 260 270 280 290 300 310 320 330 340Wavelength, (nm)

(OD

0.00

0.10

310 330 350 370 390 410 430 450 470

Wavelength (nm)

Fluo

re

(Far‐UV) Circular  Dichroism      (Near‐UV)

10000

15000

RECD 16283-10-030RECD 16283-10-037

40

50

RECD 16283-10-030

RECD 16283-10-037

RS028-001

-10000

-5000

0

5000

200 210 220 230 240 250 260

MR

E, (

nm)

RS028-001

10

0

10

20

30

260 280 300 320 340 360

MR

E, (

nm)

S0 8 00

-20000

-15000

Wavelength, (nm)-30

-20

-10

Wavelength, (nm)

9

Impact of Overlapping Signals

UV-Vis Spectroscopy Circular Dichroism

10

11

High Resolution CZE Under Conditions that Maintain the Native Structure of the Protein

Biopharmaceutical27cm 73 cm

12

1D 1H NMR Spectrum of Lysozyme (14kDa)

Lecture slide from Dr, Randall MauldinDepartment of Biochemistry and Biophysics, UNC Medicine 13

Going From 1D 1H to 2D 15N-1H NMR

1D NMR

2D NMR

Lecture slide from Dr, Randall MauldinDepartment of Biochemistry and Biophysics, UNC Medicine 14

1D 1H NMR Spectrum of a Pegylated IFN ( 23 kD P t i )

500PEG-IFN

Aliphatics

(~23 kDa Protein)

400

450

PEGBuffer

Aliphatics

250

300

350

sity

uni

t

PEG

Aromatics & peptide bonds

150

200inte

ns

H2O

0

50

100

-101234567890

1H chemical shift (ppm)

15

Assessing Quantitative HOS Comparability (Pre- vs Post-change) Using 1D 1H NMR: Local vs Global Linear Correlation g ) g

Coefficient (Using Pearson correlation coefficient)Aromatics & peptide bonds Aliphatics

RX,Y = covariance (X,Y)/([standard deviation of X]*[standard deviation of Y])

= Σ[(X(δ) - <X>)*(Y(δ) - <Y>)]({Σ(X(δ) - <X>)2}0.5)*({Σ(Y(δ) - <Y>)2}0.5)

16

Assessing Quantitative HOS Comparability (Lot to Lot) Using 1D 1H NMR: Difference NMR with 99% Confidence Limits1D H NMR: Difference NMR with 99% Confidence Limits

0.06 <(pIFN-PUR-C10-02) - (pIFN-PUR-C10-03)>Exp 99% CL

0.04

Exp 99% CLExp 99% CL

0

0.02

ed in

tens

ity u

nit

-0.02norm

aliz

e

-0.04

-10123456789-0.06

1H chemical shift (ppm)

17

(Amide) H/DX-MS: Continuous Labeling Experiment

A) Take samples at different times & stop (slow) exchange byl i t & H

D2O(1:10 to 1:20)

lowering temp. & pH

B) Measure mass change via MS

H’s at backbone amide positions

H’s & D’s at backbone amide positions

Engen & Smith (2001). Anal. Chem. 73, 256A-265A.Wales & Engen (2006). Mass Spectrom. Rev. 25, 158-170. 18

Interferon–Beta–1a (IFN)

Met62

Met117

Met36

Met1

Cys17

19

Local, Bottom-Up, H/DX-MS (Pepsin Peptide Pattern for Interferon β-1a)

1 11 21 31 41 51MSYNLLGFLQ RSSNFQCQKL LWQLNGRLEY CLKDRMNFDI PEEIKQLQQF QKEDAALTIY

61 71 81 91 101 11161 71 81 91 101 111EMLQNIFAIF RQDSSSTGWN ETIVENLLAN VYHQINHLKT VLEEKLEKED FTRGKLMSSL

121 131 141 151 161HLKRYYGRIL HYLKAKEYSH CAWTIVRVEI LRNFYFINRL TGYLRN

• MW ~23kD• 1 N-linked glycan• 1 free –SH, 4 Met• 167 residues 20

Sequence coverage > 94%

Displaying & Evaluating H/DX-MS (Using “Relative” Mass Change vs Time Plots)( g g )

21

H/DX-MS Comparison of 2 Different IFN Lots Made Using Different Culture Media (Growth Conditions)

Standard Culture Media

T = 0.17 minT = 1 minT = 10 minT = 60 minT = 240 minNew Culture Media

DI(1) = 0DI(2) = 0

22

H/DX-MS: Comparison of IFN vs Oxidized (H2O2) IFN

T = 0.17 minT 1 iT = 1 minT = 10 minT = 60 minT = 240 min

DI(1) = 187DI(2) = 143

23

Effect of Ca2+ on rFIX & FIX region of rFIX-Fc (Fusion Protein)

rFIX: with vs without Ca2

a)D

iffer

ence

(Da

Gla EGF Catalytic

2

4

6

8FIX region of rFIX-Fc: with vs without Ca2+

Da)

-4

-2

0

2

Diff

eren

ce (D

Peptide Number, i

-8

-6

0 5 10 15 20 25 30 35 40 45 50

24

rFIX vs FIX region of rFIX-Fc With & Without Ca2+g

6Without Ca2+: rFIX vs. FIX region of rFIX-Fc

8

6

8With Ca2+: rFIX vs. FIX region of rFIX-Fc

-2

0

2

4

eren

ce (D

a)

-2

0

2

4

fere

nce

(Da)

-8

-6

-4

0 5 10 15 20 25 30 35 40 45 50

Diff

e

-8

-6

-4

0 5 10 15 20 25 30 35 40 45 50D

iffDI(1) = 0DI(2) = 0

DI(1) = 0DI(2) = 0

Peptide Number, i Peptide Number, i

25

FIX sequence coverage > 90%

Sequence Coverage of the FVIII Region of rFVIII-Fc(Fusion Protein)

416 peptides covering 94% of the FVIII sequence region in rFVIII‐Fc 26

Relative Fractional H/DX Comparison PlotrFVIII Region of rFVIII-Fc (top) vs rFVIII (bottom)g ( p) ( )

nge

onal

Exc

haiv

e Fr

actio

Rel

ati

27Peptide Number, i

H/DX Difference PlotrFVIII Region of rFVIII-Fc vs rFVIII g

nce

(Da)

Diff

eren

DI(1) = 0

28

( )DI(2) = 1

Peptide Number, i

Where are We and Where are We Going in Terms of Characterizing the HOS Protein Biopharmaceutical

1. The present biophysical tool box consists of low resolution techniques

2. Nevertheless the tools have a fair amount of orthogonality – measure different physical attributes

3. Need sensitive, high precision and high resolution biophysical tools that are robust and easy to use

4. Key – find biophysical tools capable of high dispersion so signal output consists of non-overlapping bands/peaks that provide meaningful “Fingerprint Output”

5 Small difference can/will be seen by these techniques – remember protein5. Small difference can/will be seen by these techniques remember protein biopharmaceuticals are not made “Identical” rather “Highly Similar”

6. When differences are seen, are they important?

7 All d l b d th “T t lit f th E id ”7. All drug approvals are based on the “Totality of the Evidence”

8. Balanced and transparent in managing & dealing with “Risk” & “Economics”

9. Do a better job in finding & developing these better biophysical tools9. Do a better job in finding & developing these better biophysical tools

10. What is the role of industry (biopharmaceutical & instrument companies) /academia/government in doing “9”!

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Speaker Would Like to Thank and Acknowledge the Following People:g p

Damian Houde, Julie Wei & George Bou-gAssaf

Dr. John Engen and members of his group (Northeastern Univ.)

Staff at Waters

Dr. Igor Kaltashov and members of his group (UMass, Amherst)

Hemophilia Group at Biogen Idec

Special Thanks to Drs. Rohin Mhatre & Helena Madden (Biogen Idec)

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