www.diahome.org analytical comparability of a human igg1 from different manufacturing sites after...

www.diahome.org

Analytical comparability of a human IgG1 from different manufacturing sites after cell line switch and process changes: A case study

Dr. Margit Jeschke

Head Analytical R&D

Novartis Biologics

www.diahome.org

Disclaimer

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3 | 2009 DIA Comparability | Jeschke M | 4-Feb-09

Outline

Introduction Molecular Challenges with mAb1NVS Results

• Purification Process Performance• Physicochemical tests• Biological characterization• Accelerated Stabilty Study• Preclinical & PK/PD

Summary Conclusions & Discussion Acknowledgements

4 | 2009 DIA Comparability | Comparability | Jeschke M | 4-Feb-09

Dru

g p

rod

uct hPoChPoC PhIIPhII PhIIIPhIII MarketMarket

DP

Prod. Site

50mg Lyo i.v. 150mg Lyo i.v. & s.c

Basel PharmOps

Site

Cell line

Launch site

SP2/0 CHO

Major change

Type A PoC quality

Type B PoC quality

Type CPrototype

Type EFinal market qualityQualities

Dru

g s

ub

stan

ceOverview mAb1NVS Drug Substance & Product

Type DPrototype


mAb1NVS Manufacturing Changes

Changes in drug substance manufacturing process for phase I/II clinical development:

• production cell line (Sp2/0 to CHO)

• manufacturing process

• manufacturing site

• scale-up


Summary of Changes

Process 2 Process 3 Process 4 Process 5

Cell Sp2/0 Sp2/0 CHO CHO

Site

Scale 3’000L 3’000L 1’500L 13’000L

USP hTransferrin: Celliance

hTransferrin: Millipore

w/ yeastolate no transferrin

w/ yeastolate no transferrin

Harvest

DSP

Centrifugation + microfiltration

AEX CEX


AEX CEX

Microfiltration

selective red. CEXAEX


selective red. CEXAEX

Nanofiltration

at the end of purification



at early state Process 1: SP2/0


Free thiol group required for biological activity

H-CDR3H-CDR1

H-CDR2

L-CDR3

L-CDR1

L-CDR2

LC-Cys-SH

Heavy chain (HC)

Light chain (LC)

X-ray structure of the mAb1NVS Fab fragment (2.80Å resolution)

Molecular Challenges


production of biologically active antibody:

A: fermentation of active mAb, e.g. by reduction of the cystine concentration

B: modulation of activity during protein purification by treatment with reducing agents

cysteinylation interferes with antigen binding

Medium dependent biological activity

Medium A Prod.Komponenten mg/l mg/l

D-Glucose 2000 4500

Cupric sulfate (CuSO4-5H2O) 240nMFe(III)citrate 50uMNickel(II) Chloride 0.25Zinc sulfate (ZnSO4-7H2O) 225nM

Glutathione Reduced 1 5Mercaptoethanol 50uM 50uML-Cystine-2HCl 65.15 325.75

0

100

200

300

1 2 3 4 5

Tit

er

(mg

/L)

human IgGactive

108%

102%46%

17%10% active

medium: 2055 2071

0

100

200

300

1 2 3 4 5

Tit

er

(mg

/L)

human IgGactive mAB

108%

102%46%

17%10% active

medium: A Prod.


More mAb1NVS idiosyncrasies

pre

sta

ine

d M

ark

er

3‘/

85

°C

no

t h

ea

ted

2‘

/ 6

0°C

1‘

/ 8

5°C

2‘

/ 8

5°C

10

‘/ 8

5°C

10

‘/ 8

5°C

- I

A

Xo

lair

3‘/

85

°C

Xo

lair

3‘/

85

°C -

IA

IA = treated with Iodoacetamide

Intact antibodyBy- and degradation products by SDS-PAGE (non-reducing)

Disulfide scrambling


Overview mAb1NVS comparability exercise

Comparison of post-change product to pre-change product at different levels:

Com

para

bilit

y Safety and efficacy

PK/PD

Preclinical

Biological characterization

Physicochemical level

Not done

PK/PD in healthy volunteers

Tox studies in monkeys Tissue crossreactivity studies

Target binding & potency Fc Receptor binding

Comprehensive analytical characterization


Batches for analytical comparability

Two representative drug substance batches from each process were chosen to assess:

the process performance (incl. IPC) physicochemical properties biological characterization: antigen binding, Fc receptor

binding, and potency accelerated stability (6 months)

Sp2/0 P2 Sp2/0 P3 CHO P4 CHO P5

SP2/0-S06 SP2/0-S07 CHO-B07 CHO-S07


Outline

Introduction Molecular Challenges with mAb1NVS Results

• Purification Process Performance• Physicochemical tests• Biological characterization• Accelerated Stabilty Study• Preclinical & PK/PD

Summary Conclusions & Discussion Acknowledgements


Purification Process Performance

Process performance - ProtA, HCP, DNA and yields:

• The single step efficiency is different

• Overall removal successful for all process related impurities

• Single step yields are different


Test Requirement CHO-B07 A CHO-B07 B CHO-B07 C CHO-B07 D CHO-B07 E CHO-B07 F CHO-S07 A CHO-S07 B

Appearance of the solution: Turbidity

Clear to opalescent Opalescent Opalescent

Clear

Slightly opalescent

Slightly opalescent

Slightly opalescent

Slightly opalescent

Slightly opalescent

pH value 5.5 – 6.5 6.2 6.3 6.0 6.1 6.2 6.1 6.1 6.3

Assay by SEC [mg/ml]

60.0 64.9 64.6 66.0 66.6 65.2 64.4 71.6 69.4

free SH-groups [Mol/Mol]

≥ 2.0 2.6 2.6 2.5 2.5 2.5 2.6 2.3 2.2

Impurity by SDS-PAGE (reducing)

Sum of impurities ≤ 5.0 %

0.3 % <0.25 % <0.25 % <0.25 % 0.5 % 0.4 % 1.0% 1.0%

Related substances by SEC

aggregates: ≤ 5.0 % fragments: ≤ 5.0 %

0.3 % <0.1%

0.4 % <0.1%

0.4 % <0.1%

0.4 % <0.1%

0.5 % <0.1%

0.5 % 0.3%

0.3 % 0.0 %

0.2 % 0.0 %

Inhibition of IL-x release

80% - 125 % 97 % 96 % 108 % 99 % 98 % 99 % 94% 92 %

CHO HCPs Not yet defined n.p. < 1 ng/mg < 1 ng/mg < 1 ng/mg < 1 ng/mg < 1 ng/mg < 1 ng/mg < 1 ng/mg.

Protein A ELISA ≤ 5 [ng/mg AIN457] <1 <1 <1 <1 <1 <1 <1 <1

Residual DNA ≤ 8.3 pg/mg AIN457 n.p. < 2 < 2 < 2 < 2 < 2 < 2 < 2

MLT ≤ 10 [cfu/ml] n.p. <1 <1 <1 1 <1 0 0

BET ≤ 0.1 [EU/mg] < 0.0033 < 0.0033 < 0.0033 < 0.0033 < 0.0033 < 0.0033 < 0.01 0.02

Physicochemical characterization Release tests

All release requirements were met

Comparison of release data for mAb1NVS drug substance batches (CHO processes)


• Nearly all (96%/99%) peptides were identified by MS

• CHO/CHO: Comparable pattern of peaks (no new peaks, no peak missing)

• CHO/Sp2/0: Additional peaks for C-terminal variants in the CHO material + different peaks for the carbohydrate variants

Q-ToF MS: mass differences according to the carbohydrate variations

Physicochemical characterization Protein identity – reduced Pep Map and Q-ToF MS

20 40 60 80 100 120 140 Time [min]0

200

400

600

Intens.

CHO-B07

LC

147

-150

HC

224

-228

LC

209

-215

HC

420

-424

LC

185

-189

HC

345

-348

LC

151

-170

HC

59-6

5

HC

66-7

6

HC

450

-456

HC

450

-457

HC

450

-455 LC

192

-208

HC

144

-157

HC

132

-143

HC

285

-298

,H

C44

-58

HC

337

-344

HC

351

-370

HC

371

-380

LC

171

-184

HC

425

-449

HC

381

-402

HC

403

-419

LC

109

-127

HC

259

-284

LC

1-4

0

LC

128

-146

HC

233

-258

HC

233

-256

HC

299

-327

+ N

-gly

can

s

LC

41-1

08H

C1

-43

HC

158

-220

HC

158

-215

HC

371

-424

HC

66-1

43

HC

277

-143

, HC

77-1

31

20 40 60 80 100 120 140 Time [min]0

200

400

600

Intens.

LC

147

-150

HC

224

-228

LC

209

-215

HC

420

-424

LC

185

-189

HC

345

-348

LC

151

-170

HC

59-6

5

HC

66-7

6

HC

450

-456

HC

450

-457

HC

450

-455 LC

192

-208

HC

144

-157

HC

132

-143

HC

285

-298

,H

C44

-58

HC

337

-344

HC

351

-370

HC

371

-380

LC

171

-184

HC

425

-449

HC

381

-402

HC

403

-419

LC

109

-127

HC

259

-284

LC

1-4

0

LC

128

-146

HC

233

-258

HC

233

-256

HC

299

-327

+ N

-gly

can

s

LC

41-1

08H

C1

-43

HC

158

-220

HC

158

-215

HC

371

-424

HC

66-1

43

HC

277

-143

, HC

77-1

31


Quantification: NP-HPLC Q-TOF Analysis (full size mAb) Peptide mapping of N-Glycan containing peptides

Physicochemical characterization Carbohydrate heterogeneity

1,6 bG1

1,6 bG1

1,6 / 1,3 hG0M6

SP2/0-S07

bG0ΔGlcNAc

bG0

M5

bG1ΔGlcNAc 1,3 bG1

bG2 M6 M7 M8

bG2+Gal

50 60 70 80 90 100 110 Time [min]40

0

50

100

150

Intens.

1,6/1,3 bG1ΔFucCHO-S07

bG0ΔFuc

bG0

M5

1,3 bG1

bG2 M6 M7 M8

40

0

50

100

150

Intens.


Only CHO material contains bG0/1/2 ΔFucose CHO material contains less high mannose forms Only Sp2/0 material contains bG0 ΔGlcNAc, bG1 ΔGlcNAc

0

5

10

15

20

25

bG

0-[F

uc]

bG

0-[G

lcN

Ac]

bG

0

Man

5

bG

1-[G

lcN

Ac]

1,3/

1,6

bG

1-[F

uc]

1,3/

1,6

bG

1-[F

uc]

1,6

bG

1

1,3

bG

1

Man

6

bG

2-[F

uc]

1,3/

1,6

hG

0M6

bG

2

Man

7

bG

2+G

al

M8

Sp2/0-S06

Sp2/0-S07

CHO-B07

CHO-S07

Physicochemical characterization Carbohydrate heterogeneity


10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 27.7

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

min

4

3

2

1

CHO -B07

Sp2/0 - S06

CHO -S07

Sp2/0 - S07

Aggregate – AP1

Monomer

Degradation products

10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 27.7

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

min

4

3

2

1

CHO -

Sp2/0 -

CHO -

Sp2/0 -

Aggregate – AP1

Monomer

Degradation products

Physicochemical characterization Protein impurities - SEC

Same impurity profile


0712

02

0712

03

0712

04

0712

05

0712

06

0621

01

0722

03

0722

04

Uns

tain

ed M

WM

40-5

00 K

Da

Uns

tain

ed M

WM

40-5

00 K

Da

500 KDa

290 KDa240 KDa

160 KDa

116 KDa

97 KDa

66 KDa

55 KDa

40 KDa

0712

02

0712

03

0712

04

0712

05

0712

06

0621

01

0722

03

0722

04

Uns

tain

ed M

WM

40-5

00 K

Da

Uns

tain

ed M

WM

40-5

00 K

Da

500 KDa

290 KDa240 KDa

160 KDa

116 KDa

97 KDa

66 KDa

55 KDa

40 KDa

07

120

2

07

120

3

07

120

4

07

120

5

07

120

6

06

210

1 S

p2

/0

07

220

3

07

220

4

Un

sta

ine

d M

WM

40-

50

0 K

Da

HHLL

HHL

HH

HL

H

07

120

2

07

120

3

07

120

4

07

120

5

07

120

6

06

210

1 S

p2

/0

07

220

3

07

220

4

Un

sta

ine

d M

WM

40-

50

0 K

Da CHO-S07CHO-

CHO-B07CHO-

HHLL

HHL

HH

HL

H

non blocked/non-reduced blocked/non-reduced

Physicochemical characterization Protein impurities - SDS-PAGE non-red. / silver stain

CHO-B07CHO- CHO-S07CHO-


CEX (native and after treatment with carboxypeptidase B): • All batches show the rank order of abundance of charge isoforms 00 > K0 > KK

• Same charged isoforms CHO/CHO; different charged isoforms Sp2/0/CHO

Physicochemical characterization Charge heterogenity – Comparison by HPLC-CEX

Inhb

it =

Off

8.0-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,500

4

3

2

1

Sp2/0- S06

Acidic variants

CHO-B07

CHO-S07

9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0

Sp2/0- S07

0K

1K

2K

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,500

1,625

1,750

1,875

2,000mAU

min

WVL:220 nm

Acidicvariants

0K

Additional charge variant

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,500

1,625

1,750

1,875

min

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,500

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,500

1,625

1,750

1,875

2,000mAU

min

WVL:220 nm

Acidicvariants

0K

Additional charge variant

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,500

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,500

1,625

1,750

1,875

min


Far UV Near UV

Physicochemical characterization Higher order structure – Circular dichroism spectroscopy

CD spectra of the four mAb1NVS batches are comparable


Theoretical activity (%) of mAb1NVS drug substance batches based on cystamine derivatization

Sp2/0- S06 Sp2/0-S07 CHO-B07 CHO-S07

Total calculated activity [%] 96.7 97.3 96.7 97.8

Free SH group – calculated activityCystamine labeling / CEX

50

100

150

200

250

300

350

400

450

500

CHO-B07

Sp2/0 - S07

CHO-S07

Sp2/0 - S06

400

450

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 45.0-20 minmin

-

Sp2/0 -

-

-20.1 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.6

1.4

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5

min

432

1

WVL:220 nm

50%

active

inactive

active

20.1 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.6

1.4

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5

40.0

50%

active

inactive

active


Sp2/0-S06 Sp2/0-S07 CHO-B07 CHO-S07

062101 072105 071202 072203

kon [105 M-1 s-1] 1.3 ± 0.0 1.3 ± 0.0 1.3 ± 0.0 1.3 ± 0.0

koff [10-5 s-1] 2.3 ± 0.4 2.1 ± 0.1 2.4 ± 0.1 2.0 ± 0.1

KD [10-10 M] 1.8 ± 0.3 1.6 ± 0.1 1.9 ± 0.1 1.6 ± 0.1

Binding characteristics (kon and binding constant) to human target IL-x

Sp2/0-S06 Sp2/0-S07 CHO-B07 CHO-S07

062101 072105 071202 072203 Relative biological activity [%] 99 96 100 94

Affinity (Biacore)

Comparison of Potency: Inhibition of IL-x release target cells

Biological characterization Bioassys


Biacore: • comparable high affinity binding to target

• increased affinity to FcγRIIIa and FcγRIIIb for the CHO-B07 material

Sp2/0-06 Sp2/0-07 CHO-B07 CHO-S07

FcγRIa 20.1 nM 21.2 nM 22.0 nM 24.9 nM

FcγRIIa 7.4 µM 7.7 µM 6.7 µM 7.5 µM

FcγRIIIa (F158) 13.5 µM 13.5 µM 8.6 µM 14.3 µM

FcγRIIIa (V158) 6.8 µM 6.1 µM 4.3 µM 8.1 µM

FcγRIIIb 28.9 µM 29.5 µM 18.0 µM 30.3 µM

KD values for interactions of mAb1NVS with Fc receptors

Biological characterization Fc Receptor binding


Physicochemical and Biological characterization Comparability - summary

Analytical technique

Requirement

Result

CHO/CHO Sp2/0/CHO

Identity Non-reduced molecular weight by Q-ToF MS

Same molecular mass

Reduced Lys-C Pep Map Same peptides

Carbohydrates HPLC of enzymatically cleaved and derivatized glycan residues

Same carbohydrates

Bioactivity Inhibition of IL-x release Same inhibition Affinity constants for binding to target (Biacore)

Same binding constants

Binding to soluble Fc-receptors Biacore

Same binding constants


Physicochemical characterization Comparability - summary


Requirement Result

CHO/CHO Sp2/0/CHO

Protein impurities

SDS-PAGE (reduced and non-reduced) with silver staining

Comparable band pattern + no additional bands

HPLC-SEC Same impurity profile and no new component above LOQ

Soluble aggregates by DLS

Same size distribution and average hydrodynamic radii

SLS/MALLS Same average molecular mass/ same distribution


Physicochemical characterization Comparability - summary


Requirement Result

CHO/CHO Sp2/0/CHO

Charge heterogenity

CEX undigested Same charge variants CEX digested Same charge variants IEF Same isoelectric points

Free SH-group Cystamine CEX Theoretical activity ≥ 90% for each batch

Ellman’s assay 2.2 to 2.8 Mol/Mol

Higher order structure

CD spectroscopy Same spectra


storage condition

<-60°C (for comparison)

25°C / 60% RH 40°C/75% RH

6 weeks x x x

3 months - x x

6 months - x x

Short term stability program

Test performed for the head to head stability:

• SDS-PAGE

• SEC – Assay and Purity

• CEX

• Free SH groups

• Appearance / Color / pH

• Bioassay

Accelerated stability study Program and analytical Tests


Accelerated stability study Stability results 25°C - SEC

SP2/0-S06 – SP2/0-S07 – CHO-B07 – CHO-S07

0

0.5

1

1.5

2

2.5

3

3.5

4

initial 1.5 months 3 months 6 months0

0.5

1

1.5

2

2.5

initial 1.5 months 3 months 6 months

Aggregates Fragments


Accelerated stability study Stability results 25°C – SEC & red. SDS-PAGE

MWM [kDa]

light chain

heavy chain

mA

b1N

VS

ref

eren

ce

1.5% 2.0%

--

200

1169766

55

3731

22

14

6

4

200

1169766

55

3731

22

6

4

-

0721

05 S

p2/

0-S

07

(062

101

Sp

2/0-

S06

)

0722

03 C

HO

-S07

0712

02 C

HO

-B07

6 Months 25°C

062101 / Sp2/0-S06

AP3

DP3

DP1

monomer

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0-1.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

25.0

min

4

3

2

1

WVL:210 nm

071202 / CHO-B07

072105 / Sp2/0-S07

072203 / CHO-S07

AP3

DP3

DP1

monomer

Overlay of SEC chromatograms – 3M 25°C


5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0-30

100

200

300

400

500

600

700

800

900

1,000

1,100

1,200

1 - DM_080318_01 #11 [modified by mezzodo1]

min

4

3

2

1

CHO-B07 / 071202

Sp2/0 - 07 / 072105

CHO-S07 / 072203

Sp2/0 - S06 / 062101

700

800

900

1,000

1,100

1,200

1 - DM_080318_01 #11 [modified by mezzodo1]

min

4

3

2

1

samples stored for 3 months at 25°C/60% RH

Acidic variants Basic variants

0K

Accelerated stability study Stability results 3 months 25°C – CEX


Accelerated stability study Stability results 25°C – Cell based functional bioassay

40

50

60

70

80

90

100

110

initial 1.5 months 3 months 6 months

Storage time [months]

Sp2/0-S06

Sp2/0-S07

CHO-B07

CHO-S07

initial 91 100 96 94

Storage at 25°C / 60% RH

1.5 74 95 95 105

3 76 85 92 103

6 55 68 85 88

Storage at 40°C / 75% RH

1.5 62 66 76 94

3 45 45 58 71

6 26 21 62 65 SP2/0-S06 – SP2/0-S07

CHO-B07 – CHO-S07

Bioassay


Comparability – Summary & Conclusion

Process performance - ProtA, HCP and DNA: • The single step efficiencies are different especially for DNA

• Overall removal successful for all processes

Release testing: • All release requirements and additional comparability requirements met

Stability testing: • no critical observation for any stability parameter tested

Additional testing:• CHO-CHO: differences in amount of carbohydrates

• CHO-Sp2/0:

• Differences in amount and kind of carbohydrates

• Differences in binding strength to soluble Fc receptors

• Variations in charge heterogeneity

Analysis of the materials (CHO-CHO and CHO-Sp2/0) shows some differences


Differences Sp2/0 process CHO CHO

Charge variants - Δ Lys – Gly variant- remaining charge variant after Lys digest

Carbohydrates - non-fucosylated variants

Higher amounts of ΔFucose variant

Binging to soluble FcRIIIb receptor

- slightly higher binding affinity

Processes Different efficiency in process impurity removal

Slightly lower efficiency in process impurity removal

Clinical studies PhI (Ph II) (Ph II)

Tox studies No immunogenicity was detected

No immunogenicity was detected

No immunogenicity detected

PK/PD x x x

Comparability – Summary Overview


Additional studies

Tissue crossreactivity studies• Comparison of positive staining from human tissues treated with SP2/0 and CHO derived mAB1NVS

In vivo repeated dose toxicity studies• 4 week intravenous toxicity studies in cynomolgus monkeys (incl. 8 or 10 weeks recovery)

• All serum samples were analyzed for mAB1NVS by target-based and anti-idiotype competitive ELISA

PK and TK in cynomolgus monkey• pharmacokinetic single dose PK and 4 weeks tox studies in

cynomolgus monkeys (iv dosing) with SP2/0-derived material and CHO-derived material)


Comparison of PK profiles of CHO- and Sp2/0-derived mAB1NVS Comparison of target-based and anti-idiotype ELISA assays

Similar profiles obtained with CHO- and Sp2/0-derived mAB1NVS

Co

nce

ntr

atio

n (

ug

/mL

)

PK - CHO cell

-50

50

150

250

350

450

1 2 3 4 7 10 14 21 28 36 42

Time after injection in days

Animal 1-Anti ID

Animal 1-Target

Animal 2 -Anti-ID

Animal 2 -Target

PK - SP2/O cell

0

50

100

150

200

250

300

350

400

450

0.003 1 2 3 4 7 10 14 21 28 36 42

Time after injection in days

con

cen

tart

ion

(u

g/m

l) Animal 3-Anti ID

Animal 3 -Target

Animal 4 -Anti-IDAnimal 4-Target

Single dose cyno PK study (10 mg/kg i.v.)


Toxicokinetics

(SP2/0): doses: 0, 10, 30, 100 mg/kg

(CHO): doses: 0, 15, 50, 150 mg/kg

D1

iv

D8

iv

D22

iv

D15

iv

8 (SP2/0) or 10 (CHO) week recovery

D29

TK profile TK profile

2M+2F (dose groups 0 and 100/150 mg/kg)

4 weeks cyno tox studies (3M + 3F /group)


Mean concentrations of mAb1NVS in cynomolgus serum and recovery phase

recovery males

Recovery females

SP2/0 CHO

t1/2, males = 13.7 dayst1/2, females = 20.0 days

t1/2, males = 11.6 dayst1/2, females = 14.6 days

Toxicokinetics

100

1000

10000

0 91

18

2

27

4

36

6

45

6

54

7

63

9

73

1

82

1

91

2

10

04

10

96

11

86

12

77

13

69

14

61

Time (h)

mA

b1N

VS

co

nce

ntr

atio

n (

µg

/mL

)

10

100

1000

10000

0 200 400 600 800 1000 1200 1400 1600 1800 2000

Time (hours)m

Ab

1N

VS

(u

g/m

L) Animal # 404 (M)

Animal # 405 (M)

Animal # 454 (F)

Animal # 455 (F)


Are they comparable ???

„comparable does not mean identical“FDA. August 16, 2004 (Vol 69, # 157, Pages 50386ff); Scientific Considerations

Related to Developing Follow-on Protein Products


- Paul-Ehrlich-Institute (2008)

“During characterization of the drug substance a change to the glycosylation pattern was observed during the comparability exercise. Although it was demonstrated, that the difference in glycosylation obviously had no effect on the biological function, one can not exclude that there might be an immunogenic potential of the new glycans, which were identified in the CHO-derived material and therefore glycosylation should currently be part of the release specifications in order to demonstrate batch to batch consistency.”


In-depth analytical characterization and understanding of critical quality attributes is key to successful comparability

Perform risk assessment for planned changes and discuss implications upfront

Release Specs alone are inadequate to show no impact of change. Perform side-by-side analysis whenever possible

Keep retain samples of all batches and as long as possible

overload IEF / SDS-PAGE or silver stain to see low amount impurities.

Use exact same samples used for IPC analysis

Same test methods – otherwise describe differences

Explain differences well (incl. variation of methods used)

In the discussion of differences consider intended purpose

Lessons learned


ACKNOWLEDGEMENTS

Manuela SchärpfChristoph BächlerCornelius FritschGeorg HölzlSteffen PahlichIso LengwilerManuel DiezMarc HasselHui Zhao Gerard BruinMarkus BlümelMichaela DehioYuan Xu


Novartis Biologics, Basel, Switzerland

Basel

Novartis

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