Critical Raw Material Control in Drug Substance and its Impact on Drug Product Comparability

Emerging Strategies in Drug Product Comparability and Process ValidationJuly 18 – 19, 2016, Gaithersburg, MD USA

Presented by Eran J. Benjamin, Ph.D. Bio-Product R&D, Eli Lilly and Company

Bio-Product R&D, Eli Lilly and Co.

CASSS CMC Strategy Forum

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♦ LP541 is a PEGylated Protein intended for Diabetes therapy♦ Presentation:

♦ The drug substance is a frozen solution (≤-10°C)♦ The drug product is a refrigerated multi-use product (2-8°C)

Drug Product

(Refrigerated)Cartridges & Vials

~23 mg/mL

Drug Substance Purification and

Isolation(Frozen Solution)

~ 45 gram/Liter

Purified Protein Intermediate

(Bulk Powder)

PEGylation reagent

(Bulk Powder)

Buffer SectionCompendial Excipients

Program Overview

♦ 20 kDa PEG uses Paranitrophenyl carbonate activation♦ Specific to a Lysine side chain residue♦ Generates a stable Urethane bond

Structural Formula (n = approx. 450 subunits):

♦ It defines the time-action profile that the patient would experience

♦ To allow commercialization, 3 suppliers were needed♦ However:

♦ PEG is polymeric distribution♦ Each supplier manufactures PEG with slightly different quality attributes

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20 kDa PEG is a critical raw material

PEG Mw (weight average molecular weight)

♦ Molecular weight (Mw) is a critical quality attribute of PEG♦ Mw is defined as the weighted average♦ PEG suppliers were using GCP to measure Mw

Mw (Weighted Average Molecular Weight)

Mass analysis of 20 kD PEG sample

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PEG Polydispersity Index (PDI)

♦ The breadth of the distribution is also a Critical Quality Attribute♦ The term for polymeric distribution is: Polydispersity Index (PDI)

0

0.1

0.2

0.3

0.4

0.5

0.6

Nor

mal

ized

Heig

ht

Mn, Number Average MW

Normalized Mn Curves for 20 kDa Mw PDI = 1.003 (Representative of all CT Materials) & PDI = 1.05 (GPC Spec Limit)

PDI 1.003

PDI 1.05

PDI = 1.003Clinical Experience

PDI = 1.05 (PEG suppliers)Far removed from clinical experience

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PEG Mw and PDI and its impact to PK

♦ Extreme Mw could effect PK (Rodent Model)♦ PEGylated samples with various PEG size♦ 5 kD and 10 kD mPEG significantly different♦ Supplier range acceptable: 18-22 kD♦ Combination (intentionally increasing PDI) acceptable in 18-22 kD range

Rat IV clearance study

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Phase 2 to Phase 3 PEG supply

♦ Map of PEG of suppliers: • Parameters with potential to impact Drug Product CQAs

Test Supplier 1 (Phase 1/2 and 3)

Supplier 2 (Phase 3)

Supplier 3 (Phase 3)

Weight Average Molecular Weight (MW, Da) - GPC 18,000 – 22,000 18,000 – 22,000

Number Average Molecular Weight (MN, Da) - GPC 18,000 – 22,000 Report

Polydispersity Index NMT 1.10 NMT 1.05 NMT 1.05

Bi-functional PEG (%)* NMT 3 NMT 4

20 kDa Fraction (%) NLT 90

Functional Activity (%) NLT 80 NLT 80 NLT 90

Free p-Nitrophenol (ppm) NMT 200 Report

7* PNP-PEG-PEG-PNP Bi-functional PEG – has the potential to generate covalent dimers

Phase 3 Mw Data

S

0High resolution LC-TOF-MS data:♦ Data collected on PEG used in Phase 3 supply♦ Each Supplier is able to reproduce Mw♦ However; statistically significant differences are observed between suppliers

Supplier 2

Supplier 3 Supplier 1(Phase 1/2)

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Phase 3 PDI Data

High resolution LC-TOF-MS data:♦ Data collected on PEG used in Phase 3 supply♦ Each Supplier is able to control PDI♦ Populations are similar amongst different suppliers

Supplier 2 Supplier 3 Supplier 1(Phase 1/2)

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PEG attributes that may affect DP CQAs

LP541 Degradation Pathways

Dimer and HMWP formation

De-PEGylationor PEG degradation

GlutamineDeamidation(pH induced)

Disulfide Rearrangements

(occurs mainly in API)

Could PEG properties influence these CQAs ?

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Analytical comparability study

♦ A comparability assessment was conducted at the start of Phase 3• Examined PEG raw materials• Multiple parameters related to drug substance and drug product

♦ Drug substance testing:1. Release testing comparison2. Characterization and biophysical testing3. Accelerated stability studies / cell based bioassay

♦ Drug product testing:1. In-process solution testing 2. Release testing comparison3. Characterization testing4. Accelerated stability studies / cell based bioassay

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SEC Analysis / DP Demo Batches

SEC% HMWP

PEG Storage condition T=0 0.5 M 1M 2M 6M

Supplier 12-8 °C

30°C/60%RH40°C/75% RH

1.2

2.61.6 2.2

1.4

Supplier 22-8 °C

30°C/60%RH40°C/75% RH

1.4

2.71.9 2.5

1.6

Supplier 32-8 °C

30°C/60%RH40°C/75% RH

1.6

3.01.9 2.8

1.8

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AU

-0.020

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

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HMWP

LP541 Monomer Metacresol

6 Month StressedDrug Product Sample@ 30°C/65%RH

RP-HPLC Analysis / DP Demo Batches

RP-HPLC Related Substances

and Impurities: % Total

PEG Storage Condition T=0 0.5 M 1M 2M 6M

Supplier 12-8 °C

30°C/60%RH40°C/75% RH

1.3

3.12.3 2.5

1.5

Supplier 22-8 °C

30°C/60%RH40°C/75% RH

1.4

3.22.3 2.9

1.7

Supplier 32-8 °C

30°C/60%RH40°C/75% RH

1.6

3.62.7 3.2

2.0

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AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

LP541 Metacresol

DimersHigher Order

Multimers

Proteinfragments

6 Month StressedDrug Product Sample@ 30°C/65%RH

SEC and RP-HPLC Analysis: Phase 3 DP

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♦ Monitoring of comparability continues throughout program♦ Phase 3 batches shown:

• Lower Mw PEG (supplier 3), influences:– API purification parameters– Results in reduced purity (may be compensated by column load)

• Stability performance remains constant.

PEG remains intact over the shelf-life

DS Batch C065137Initial

DS Batch C0651379 weeks, 25°C, 60 % RH

DP Batch TP12089Initial

DP Batch TP120893 Months, 30°C, 65% RH

♦ Mass analysis of stressed drug substance and drug product

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PEG remains intact over the shelf-life

♦ Expanded Plot of [M+5H]5+ TOF-MS Spectra♦ Identical observations for all 3 suppliers

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CEX Analysis

CEX-HPLC Related Substances

and Impurities: % Total

PEG Storage Condition T=0 0.5 M 1M 2M 6M

Supplier 12-8 °C

30°C/60%RH40°C/75% RH

4.8

5.95.3 5.9

3.7

Supplier 22-8 °C

30°C/60%RH40°C/75% RH

5.5

6.66.2 6.6

3.7

Supplier 32-8 °C

30°C/60%RH40°C/75% RH

5.1

6.36.0 6.7

4.0

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.042

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1 2 3

46 7 5

Dea

mid

atio

nC

lip

N-te

rmin

us P

EG

Disulfide rearranged impuritiesD

i-PEG

ylat

ion

High stress sample

System suitability

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CEX Analysis: Phase 3 DP

1835689C 0356

SS

S

S

♦ Monitoring of comparability continues throughout program♦ Phase 3 batches shown

• Stability performance remains constant

Peptide Map

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AU

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00

♦ Glu-C peptide map characterization method• PEGylated fragment is late eluting (not shown)• Utilized to confirm CEX-HPLC observation• Sensitive to deamidation and disulfide rearrangements

Metacresol

PeptidefragmentsPeptide

fragments

Bioassay as a potential CQA

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70.0

80.0

90.0

100.0

110.0

120.0

130.0

0 3 6 9 12

Pote

ncy B

y Cell

Bas

ed B

ioas

say (

%)

Time (Weeks)

C082449

C096546

C096548

C141565

C146588

C146591

C153910

C158833

Phase 3 API (PS + PV)Stressed at 25°C:

♦ 3 PEG suppliers♦ Purity decline observed by

RP-HPLC ♦ Potency decline is difficult

to detect beyond method variability 70.0

75.0

80.0

85.0

90.0

95.0

100.0

0 3 6 9 12Pu

rity

RP-H

PLC

(%)

C082449

C096546

C096548

C141565

C146588

C146591

C153910

C158833

Bioassay Drug Product Data

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70

80

90

100

110

120

130

0 1 2 3 4 5 6

Pote

ncy

by C

ell b

ased

Bio

assa

y (%

)

Time (Months)

C142266

C142474

C146136

70

75

80

85

90

95

100

0 1 2 3 4 5 6

Puri

ty R

P-H

PLC

(%)

Time (Months)

C142266

C142474

C146136

Phase 3 DP PS batchesStressed at 30°C:

♦ Each batch is different PEG supplier

♦ Purity decline observed by RP-HPLC

♦ Potency decline is difficult to detect beyond method variability

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Phase 3 accumulated experience

♦ 13 commercial scale API batches (data shown)♦ 11 drug product batches♦ The data set allows applying statistic tools for comparability studies

Clinical Evaluation of PEG Suppliers

♦ Phase 1 PK/PD Study♦ Healthy volunteers administered single injection of LP541 with each PEG

supplier on 3 separate occasions followed by PK sampling♦ CMC provided information on CT batches including the Mw of the PEG

batches used♦ No correlation was observed to PEG Mw♦ No difference observed in PD

♦ Phase 3 Study (one of the global Phase 3 studies)♦ Patient population♦ Open label study, LP541 arm randomized to a single PEG supplier ♦ Subgroup analysis as a function of PEG supplier♦ Different suppliers did not impact the PK and PD♦ No difference in safety data

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PEG Supplier Evaluation

Clinical Efficacy/Safety

PK/PD (human)

ToxicologyStudies

Release & Stability Testing

Bioassay Testing

Additional Characterization Testing

Phase 2/3 PV and BLA

Primary Stability:Statistical comparability compared to clinical experience to date(API site change)

Start of Phase 3:Side by Side PEG Supplier comparability analytical data package

Phase 3 Global StudyPhase 1 PK/PD Study

Mixed PEG:Analytical comparability demonstrated with mixed PEG suppliers to support commercial supply chain flexibility

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Control Strategy for Commercial Supply

♦ For commercial supply:• Drug substance was stored frozen in 50 L HDPE containers• Required a “floating batch” strategy for DP

♦ The DP facility was intending on using a FIFO approach

• LC-TOF analysis for Mw and PDI control the incoming PEG reagent

• RP-HPLC and CEX-HPLC ensures that only one PEG is attached to protein and at the correct position

• RP-HPLC ensures that de-PEGylation does not occur over shelf-life and patient in-use period

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PEG

• Mw• PDI

Drug Substance

• RP-HPLC• CEX-HPLC

Drug Product

• RP-HPLC

Quality control of the drug productA

U

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

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0.22

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0.26

0.28

0.30

0.32

0.34

0.36

0.38

0.40

0.42

0.44

0.46

0.48

0.50

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00 60.00 62.00 64.00

CEX Chromatography of DP made with more than one source of PEG-API

1.7 kD MW difference - OKLot H01020-121-A18.4 kD + 20.1 kD PEG

2.3 kD MW difference - OKLot H01020-121-B20.1 kD + 22.4 kD PEG

3.1 kD MW differenceLot H01020-121-E18.4 kD + 21.5 kD PEG

4.0 kD MW differenceLot H01020-121-C18.4 kD + 22.4 kD PEG

• If two PEG batches are combined that are separated by more than 2.3 kD, failures in the analytical methods would occur.

• Also occurs in SEC & RP-HPLC.• Controlling this by segregating

PEG in DS manufacturing is complex.

• Note: 18.4kD and 22.4kD PEGs intentionally ordered from suppliers.

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Statistical Model for Mw and applying a singular control limit across suppliers

Supplier 2 3 1 (Phase 1/2)N= 17 7 22Average Mw 21727 20125 21484

Max Mw 21871 20324 21716

Min Mw 21585 19880 21335

Std. Dev. 88 145 74± 6 Std. Dev (rounded) 21200 - 22260* 19260* - 20990 21040 - 2193095/99.5 Tolerance levels 21364 - 22089 19299 - 20950 21199 - 21768

18,000 19,000 20,000 21,000 22,000

TOF-MS specification 19,260-22,260 Dalton

Supplier 2Supplier 3

Supplier 1Predicted TOF-MS with ±6 Std. Dev.

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Proposed commercial PEG specifications

Test Procedure Acceptance criteria Critical parameter

Physical Appearance Visual Appearance White to Off-white Powder

Identity LC-TOF-MS Retention time cGMP requirement

Weight Average Molecular Weight LC-TOF-MS NLT19.3 kDa and

NMT 22.3 kDaImparts the long-acting PK/PD profile

Polydispersity Index LC-TOF-MS Not more than 1.01 Defines distribution of molecular weights

Functional Activity RP-HPLC Not less than 90 % Impacts only at extreme conditions)

Bi-functional PEG RP-HPLC Not more than 4.0 % Shown not to induce dimers

Water Content Karl-Fischer Not more than 1.0 % Significant hydrolysis could impact reaction

Free p-Nitrophenol RP-HPLC Not more than 200 ppm No known impact

Residual Solvents GC Review of Supplier Certificate of Analysis

Considerable process removal exists

♦ A statistical comparison of drug product accelerated stability data (30°C/65% RH) was conducted.

♦ Intent was to verify the comparability of drug product manufactured using different PEG suppliers versus mixed PEG suppliers.

♦ Acceptance criteria were applied to batch release and characterization data

♦ Acceptance criteria were applied to accelerated stability data.♦ Mixed PEG source DP was generated from all 3 possible

combinations:• Supplier 1 + Supplier 2• Supplier 1 + Supplier 3• Supplier 2 + Supplier 3

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Introducing mixed PEG supplier batches

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♦ Common slope test:• A singular slope is provided for the mixed PEG supplier• When the p-value is greater than 0.05, no statistical differences exist• p-Value was 0.69

Introducing mixed PEG Supplier batches

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♦ Common slope test:♦ p-Value was 0.046 and required further assessment:

• The mix PEG supplier data had a slightly lower degradation rate indicating that using mix PEG suppliers would be acceptable

• As more data was collected, the apparent differences diminished

Introducing mixed PEG Supplier batches

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Introducing mixed PEG Supplier batches

♦ Common slope test:• A singular slope is provided for the mixed PEG supplier• When the p-value is greater than 0.05, no statistical differences exist• p-Value was 0.59

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Summary

♦ Analytical comparability (including in-vitro data) was used to introduce two PEG suppliers into the Phase 3 program

♦ PEG methods and control parameters were refined to allow a viable commercial supply chain using 3 PEG suppliers

♦ Analytical comparability with statistical acceptance criteria was utilized to allow mixed PEG supplier batches (Drug Product)

♦ Clinical Comparability was demonstrated during the Phase 3 registration program

Acknowledgements

♦ Team Members:♦ Cindy Payne♦ Stan Szewczyk♦ Chi Nguyen

♦ Characterization:♦ Clayton Gough

♦ Purification Scientist:♦ Dr. Jonathan Webb

♦ Statistician:♦ Kevin Guo

♦ TS-MS Senior Engineer:♦ Andrew Rockabrand

♦ RACMC Consultants:♦ Allison Wolf♦ John Dougherty♦ Dr. Bruce Meiklejohn

♦ Bio-Product Consultant:♦ Dr. Viren Sarin

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