Comparison of Host Cell Protein

Profiles Between an Innovator and

a Biosimilar Monoclonal Antibody

Catalin E. Doneanu, Ph.D.

Waters Corporation

May 19th, 2016

2016 Waters Corporation 1

 Outline

Introduction of the 2DLC-HDMSE assay for identification and

quantification of low-abundance host cell proteins (HCPs) from
high-purity mAbs

Results of the Innovator vs Biosimilar comparison (Infliximab vs

Inflectra)

Recent results from a 1D LC-HDMSE assay of spiked Inflectra

Fast HCP monitoring assay using Progenesis QIP

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 Purification of therapeutics mAbs

Expression in CHO cell culture (~ 50% mAb, 50% HCPs)

Protein A purification (98-99% mAb)

Cation exchange chromatography (99.9% mAb)

Anion exchange filtration

Formulation (20 mg/mL typical mAb concentration)

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 Waters 2D LC-MS Platform for HCP Discovery
Assay

Integrated technologies that address the HCP Discovery LC-MS workflow:

Xevo SYNAPT
G2-XS G2-Si
Columns
CSH 300 m

Standards
OR
Informatics

MSE MSE & HDMSE

LC MS
ACQUITY UPLC QTof Mass
M-Class System Spectrometry
with 2D Technology

Training & Support

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 Diagram of the 2D-LC HCP setup

XBridge Peptide
BEH C18 300,
ACQUITY UPLC M-Class
5mm, 1.0 x 50mm
Symmetry C18 2D HCP Trap
column
Column, 5mm, 300mm x
25mm
ACQUITY UPLC M-Class
CSH Column, 1.7mm, 130,
300mm x 150mm

MS

WASTE

BSM 10 L/min

BSM1 10 L/min

mAbs, 2012, 1, 24-44

Anal Chem, 2015, 87, 10283-10291

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 Diagram of the Synapt G2-Si instrument

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 The role of precursor level ion mobility
separation

without IM separation

HCP peptide with IM separation

Anal Chem, 2015, 87, 10283-10291

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 Comparison of MS/MS spectra of the HCP
peptide zoom view I
fragment m/z = 494.19 (b4+1)
without IM separation

with IM separation

pure MS/MS spectrum

Anal Chem, 2015, 87, 10283-10291

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 Comparison of MS/MS spectra of the HCP
peptide zoom view II
fragment m/z = 631.33 (y11+2)
without IM separation

with IM separation

pure MS/MS spectrum

Anal Chem, 2015, 87, 10283-10291

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 Comparison of MS/MS spectra of the HCP
peptide zoom view III
fragment m/z = 1261.65 (y11+1)

without IM separation

with IM separation

pure MS/MS spectrum

Anal Chem, 2015, 87, 10283-10291

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 Diagram of the Synapt G2-Si instrument

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 2D-LC ESI-IM-MS/MS spectrum of the
HCP peptide
HCP: NSFL1 cofactor p47, average measurement: 7 ppm

2D Fraction: 3/10

A Precursor m/z = 877.92 (+2)

CE = 42 V
S Y Q D P S N A Q F L E S I R
y11
a2
[y11]+2

[y4-NH3]+2

y12
Q b4
[y13-H2O]+2 y7
y3
y4 y8
[y12]+2 y10
y9
y13

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 Complete list of HCPs identified and
quantified in the NIST mAb 2D HDMSE data

Anal Chem, 2015, 87, 10283-10291

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 Outline

Introduction of the 2DLC HDMSE assay for identification and

quantification of low-abundance host cell proteins (HCPs) from
high-purity mAbs

Results of the Innovator vs Biosimilar comparison (Infliximab vs

Inflectra)

Recent results from a 1D LC-HDMSE assay of spiked Inflectra

Fast HCP monitoring assay using Progenesis QIP

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 How to assess similarity between
Innovator and Biosimilar products?
Comparison of:

1) Intact or subunit ESI-MS spectra

2) LC bioseparations (1 D or 2D) of intact/protein subunits (IEX, SEC, HIC, etc)

3) 1D LC-MS peptide maps

4) Disulfide bond mapping

5) Glycan profiles following glycan cleavage,

derivatization and 1D LC-FL/MS analysis

6) Identification of sequence variants

5) HCP profiles

6) HDX

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 Comparison of HCP Profiles between
Infliximab and Inflectra

INFLIXIMAB

INFLECTRA

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 HCP Peptide WEVAELR detected in the
Infliximab digest
Fraction 4/10

Rep01

Rep02

Rep03

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 HCP Peptide WEVAELR detected in the
Inflectra digest
Fraction 4/10

Rep01

Rep02

Rep03

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 HCP Confirmation: MS/MS spectrum of
HCP peptide WEVAELR
HCP: Epidermal growth-factor like protein 8, average measurement: 15ppm

2D Fraction: 4/10 y4 Quad isolation window: ~ 2Da

CE = 22 V

W E V A E L R

y5

a2 y3
b3-H2O
b2-H2O
y1
b2

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 HCP Peptide AICQLVK detected in the
Infliximab digest
Fraction 4/10

Rep01

Rep02

Rep03

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 HCP Peptide AICQLVK detected in the
Inflectra digest
Fraction 4/10

Rep01

Rep02

Rep03

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 HCP Confirmation: MS/MS spectrum of
HCP peptide AICQLVK
HCP: WD repeat containing protein 37, average measurement: 25ppm

precursor (+1)
2D Fraction: 4/10
Quad isolation window: ~ 2 Da

CE = 36 V

A I C Q L V K

b6
b5
a5
y3
b4 a6

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 Full Sequences of Infliximab/Inflectra
HCPs

>sp|Q6GUQ1|EGFL8_MOUSE Epidermal growth factor-like protein 8

MGLWAELCISLRGLSFFLVLMTGEGTRGGSFKESLGVCSKQTLLVPLRYNESYSQPVYKPYLTLCAGRRICSTYRTTYRVAWRE
VRREVPQTHVVCCQGWKKPHPGALTCDAICSKPCLNGGVCTGPDRCECAPGWGGKHCHVDVDECRASLTLCSHGCLNT
LGSFLCSCPHPLVLGLDGRTCAGGPPESPTSASILSVAVREADSEEERALRWEVAELRGRLEKLEQWATQAGAWVRAVLPM
PPEELRPEQVAELWGRGDRIESLSDQVLLLEERLGACACEDNSLGPSLRG

>sp|Q8CBE3|WDR37_MOUSE WD repeat-containing protein 37

MPTESGSCSTARQAKQKRKSHSLSIRRTNSSEQERTGLPREMLEGQDSKLPSSVRSTLLELFGQIEREFENLYIENLELRREID
TLNERLAGEGQAIDGAELSKGQLKTKASHSTSQLSQKLKTTYKASTSKIVSSFKTTTSRAICQLVKEYIGHRDGIWDVSVTRT
QPIVLGTASADHTALLWSIETGKCLVKYAGHVGSVNSIKFHPSEQLALTASGDQTAHIWRYVVQLPTPQPVADTSQQISGED
EIECSDKDEPDIDGDVSSDCPTVRVPLTSLKSHQGVVIAADWLVGGKQVVTASWDRTANLYDVETSELVHSLTGHDQELTH
CCTHPTQRLVVTSSRDTTFRLWDFRDPSIHSVNVFQGHTDTVTSAVFTVGDNVVSGSDDRTVKVWDLKNMRSPIATIRT
DSAINRINVCVGQKIIALPHDNRQVRLFDMSGVRLARLPRSSRQGHRRMVCCSAWSEDHPICNLFTCGFDRQAIGWNIN
IPALLQEK

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 Summary of the HCP Comparison

The same 2 HCPs were detected in both mAbs (Infliximab and

Inflectra)

The HCP levels for the biosimilar mAb were 2-4 fold higher

The 2D LC-HDMSE assay can distinguish minute differences in

the HCP profiles between innovator and biosimilar mAbs

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 Outline

Introduction of the 2DLC HDMSE assay for identification and

quantification of low-abundance host cell proteins (HCPs) from
high-purity mAbs

Results of the Innovator vs Biosimilar comparison (Infliximab vs

Inflectra)

Recent results from a 1D LC-HDMSE assay of spiked Inflectra

Fast HCP monitoring assay using Progenesis QIP

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 Dependence of Peak Capacity on the
Gradient Length for Peptide Separations
Peak
capacity

Gradient length Peak

(min) capacity

5 48 90 min gradient
10 97
15 127
30 223
45 292
60 341
90 421
180 542
360 604

Gradient length (min)

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 BPI Chromatogram
Peak Width Measurement Inflectra peptide

HC T12: TEDTGVYYCSR

8.2 sec 10%

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 BPI Chromatogram
Peak Width Measurement ENL T43 peptide

8.0 sec 10%

ENL T43: VNQIGTLSESIK

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 Precursor level Ion Mobility Separations
ENL T43

Base peak ion mobilogram

Extracted mass mobilogram for ENL T43 precursor: 644.86 (+2)

(ms)

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 ESI-MS Spectra of ENL T43 Precursor Level
(A) MSE and (B) HDMSE
No IM separation

A
*
A

with IM separation
B
T43

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 ESI-MS/MS Spectra of ENL T43
(A) MSE and (B) HDMSE Enlarged View I

without IM separation

y8

with IM separation

y8

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 ESI-MS/MS Spectra of ENL T43
(A) MSE and (B) HDMSE Enlarged View II

without IM separation

y9

with IM separation

y9

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 ESI-MS/MS Spectra of ENL T43
(A) MSE and (B) HDMSE Enlarged View III

without IM separation

y10

with IM separation

y10

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 Protein Concentrations Expressed in ppm
measured in the MIX-4 spiked Inflectra digest

HDMSE dataset

Protein Molecular Amount on-column Protein conc RSD

ID weight (kDa) femtomoles nanograms ng/mL ppm (ng/mg) (%)

ADH 36.7 1091 40.0 160.2 267 3.6

PHO 97.1 1000 97.1 388.4 647 0.0

BSA 66.3 242 16.0 64.2 107 3.4

ENL 46.6 42 2.0 7.8 13 4.2

Protein loading amounts were: - 1,000 fmoles ADH

- 1,000 fmoles PHO
- 250 fmoles BSA
- 50 fmoles ENL

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 Outline

Introduction of the 2DLC HDMSE assay for identification and

quantification of low-abundance host cell proteins (HCPs) from
high-purity mAbs

Results of the Innovator vs Biosimilar comparison (Infliximab vs

Inflectra)

Recent results from a 1DLC HDMSE assay of spiked Inflectra

Fast HCP monitoring assay using Progenesis QIP

2016 Waters Corporation 35

 Tracking ADH peptides

Expected fold change: 5X Measured fold change: 5.5 X RSD: 21.7%

(7 peptides)

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 Tracking ENL peptides

Expected fold change: 5X Measured fold change: 4.5 X RSD: 11.7%

(15 peptides)

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 Tracking PHO peptides

Expected fold change: 3X Measured fold change: 3.4 X RSD: 17.1%

(17 peptides)

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 Example of HCP tracking in Progenesis
ENL T43 peptide trend plot

Blank 10 fmoles 50 fmoles 100 fmoles 250 fmoles 500 fmoles

In this experiment, all 4 proteins standards (ADH, BSA, PHO and ENL) were spiked at the same
concentration level in the Infliximab digest.

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 Correlation Plots measured vs spiked
concentrations
Measured conc

ENL

Spiked conc

Measured conc
Measured conc

PHO ADH

Spiked conc Spiked conc

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 Waters HCP Solution: HCP Identification and
Quantification with 1D and 2D Platforms

HCP Identification Levels: Can track HCP levels ~5x below these levels

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 To learn more about LC-MS Based Assays for
HCPs, please visit:
www.waters.com/hcptoolbox

A presentation with scripted

notes that you can customize to
talk about the benefits of LC-MS
in HCP analysis

Biotherapeutic

A collection of papers highlighting

the benefits of adopting LC-MS in
HCP analysis

HCPs

Supporting documentation that

describes the value of choosing
Waters LC-MS solutions for HCP
analysis
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 Acknowledgements:

Malcolm Anderson
Brad Williams
Ying Qing Yu
Weibin Chen
Asish Chakraborty
Scott Berger
Alain Beck

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