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Unprocessed C-terminal Lys variant observed only in the Biosimilar 1 peptide map.

INTRODUCTION Biopharmaceutical companies are challenged to design efficient analytical strategies for detailed assessment of structural comparability between biosimilar and innovator products. Extensive characterization increases confidence that a biosimilar product is safe, and will meet regulatory compliance requirements for abbreviated approval pathways. Here, we d emons t ra t e h ow an i n t e g ra t ed biopharmaceutical LCMS system utilizing the UNIFI Scientific Information System addresses these challenges by integrating and automating data acquisition, data processing, and result reporting into a seamless workflow for in-depth biotherapeutic LC/MS characterization.

In this study, comparability between an innovator Rituximab monoclonal antibody (mAb) and two candidate biosimilars was performed at the levels of intact protein and peptide mapping LCMS analysis. Differences in Critical Quality Attributes such as primary structure (mutation), glycan fucosylation, and and terminal amino acid heterogeneity were observed.

COMPLEMENTARY LCMS METHODOLOGIES CAN IDENTIFY CRITICAL QUALITY ATTRIBUTE DIFFERENCES BETWEEN INNOVATOR AND BIOSIMILAR RITUXIMAB

Vera B. Ivleva, Ying Qing Yu, Hongwei Xie, Sean M McCarthy, Scott J. Berger, Weibin Chen Waters Corporation, 34 Maple St, Milford MA 01757

METHODS Sample Preparation

Intact Mass Analysis Innovator and both Biosimilar mAb samples were diluted to 0.5 mg/mL in 25 mM ammonium biocarbonate. Reduced mAb Analysis The samples were diluted to 1mg/mL in a reduction buffer (25 mM NaCl, 25 mM Tris, pH 7.5) and incubated in 1mM DTT at 37oC for 20 min. The reduced samples were further diluted in 5% acetonitrile, 0.1% TFA to 0.2 mg/mL for analysis. Protein Digestion The samples were mixed with a denaturing buffer (8M guanidine chloride, 1M Tris, pH 7.5) to 1mg/mL, reduced with 3 mM DTT, and alkylated with 7 mM iodoacetomide before buffer exchange to a digestion buffer of 100 mM Tris, pH 7.5. The samples were digested individually using either Trypsin or Chymotrypsin (S:E = 20:1) for 4 hrs. The digested samples were diluted with 3% acetonitrile, 0.1% TFA to 0.2 mg/mL for injection.

Biopharmaceutical System Solution with UNIFI (Fig. 1)

ACQUITY® UPLC H-Class with PST and PrST UPLC Chemistries XEVO® G2 Tof, TUV Optical detector UNIFI v1.5.1 Scientific Information System

DATA PROCESSING AND REPORTING RESULTS

CONCLUSIONS

UPLC/TOF MS analysis at Intact mAb, Reduced mAb, and peptide map levels enabled the detection of primary structural differences, and quantitative assessments of structural variation.

Integrated and automated data acquisition, processing, and reporting for multiple analytical workflows enabled the efficient assessment of critical product attributes with minimal manual intervention.

The K R mutation detected for a candidate biosimilar of Rituximab is not readily detectable under tryptic digest analysis, and demonstrates the utility of routinely employing alternative digestion enzymes for product characterization.

MSE data of a chymotryptic digest reveal a single amino acid substitution (K R) in Biosimilar 2

Figure 1. Biopharmaceutical System Solution with UNIFI

INTACT PROTEIN ANALYSIS

REDUCED PROTEIN ANALYSIS

PEPTIDE MAP COMPARISON

REVEALING C-TERM LYS VARIANTS

PEPTIDE MAP COMPARISON REVEALING AMINO ACID MUTATION

MaxEnt-1 deconvoluted mass spectra

The following examples are measured and compared among Innovator and both Biosimilar mAbs: C-terminal Lys variants on the HC Glycoform variant (G0) between biosimilars Amino acid sequence variant (+28 Da) on the

heavy chain of Biosimilar 2 N-terminal pyroglutamination Q (PyrQ) levels

UNIFI Method Setup Components plots in compare mode

Chymotryptic Map Components plots in compare mode

m = 28 Da

Innovator

Biosimilar 2

Chymotryptic coverage of the Biosimilar 2 heavy chain

K218→ R218

MSE spectrum of the highlighted peptide with amino acid substitution

Innovator

Biosimilar 2

Tryptic Map BPI mass chromatograms in compare mode

Tryptic digest does not s h o w s i g n i f i c a n t differences between Innovator and Biosimilar 2 samples

Innovator

Biosimilar 1

G0F/G1F

G0F/G0F

G1F/G2F

G2F/G2FG0/G0F

G1F/G1FG0F/G2F

Summary plot of LC PyrQ N-terminus

Biosimilar 2 glycoforms have a systematic mass shift of 56 Da with respect to the innovator mAb

MaxEnt-1 deconvoluted mass spectra in compare mode

Distribution of G1F with C-terminal Lys variation

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

1 2 3 4 5 6 7 8 9

MS Response

Innovator

Biosimilar 1

Biosimilar 2

Distribution of G0

MS Response

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

1 2 3 4 5 6 7 8 9

InnovatorBiosimilar 1

Biosimilar 2

Summary

plots based

on UNIFI results

Innovator

Biosimilar 2

G0F/G1F

G0F/G0FG1F/G1FG0F/G2F

G1F/G2F

G2F/G2FG0/G0F

∆m = 56 Da

Innovator

Biosimilar 1

G0F G1F

G2FG0F+K G1F+KG0

G0

G0F

Innovator

Biosimilar 2

G0F G1F

G0F+K G2FG1F+K

G0

G0

G0F

G1F

∆m = 28 Da

Innovator

Biosimilar 1