TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation

INTRODUCTION The complexity of biopharmaceutical molecular entity places great challenges to the biopharmaceutical industry to manufacture safe and effective biosimilar products. Analytical methods including intact mass analysis, subunit mass analysis (e.g. light and heavy chains of mAbs), peptide mapping and released glycan profiling are frequently employed to generate valuable information to guide the development of biological drugs. In this study, we describe a LC-MS workflow using Waters biopharmaceutical system solution with UNIFI Scientific Information System to characterize and screen a recombinant monoclonal antibody (mAb), Infliximab, which was harvested from two cell lines (CHO and SP2/0).

The analysis is focused on the subunits of Infliximab (light chain and heavy chain), and structural variation comparison is made based on summary statistical results from the analysis of multiple batches of products.

Unifi scientific information system enables automated trending and summary statistical calculations so rapid data review and efficient reporting of the key results from this study is obtained.

ASSESSING STRUCTURAL VARIATION OF INFLIXIMAB PRODUCED BY TWO CELL LINES FOR BIOSIMILAR DEVELOPMENT

USING WATERS BIOPHARMACEUTCAL SYSTEM SOLUTION WITH UNIFI

Henry Shion, Vera Ivleva, Weibin Chen Late Stage Development, Pharmaceutical and Life Science, Waters Corporation, Milford, MA, USA

Figure 1. Biopharmaceutical System Solution with Unifi.

Buffer solution for mAb reduction

A reduction buffer solution containing 25 mM NaCl, 25 mM Tris, 1 mM EDTA (pH 8.0) was made

to prepare mAb subunits by reducing Infliximab with DTT.

Reduction of Infliximab

Formulated mAb solution (21.0 mg/mL, the commonly used concentration level for patient

injection) was diluted to a concentration of 1.0 mg/mL. Dithiothreitol (DTT) at 100 mM (in H2O)

was added to make final DTT concentration of 1.0 mM for reduction (incubated with dry air bath

at 37ºC). At 20 min, the sample was diluted 10 folds using an aqueous solution containing 3%

Acetonitrile and 0.1% Formic acid. This sample was then injected for LC/MS analysis of mAb

subunit.

Biopharmaceutical System Solution with UNIFI

ACQUITY UPLC H-Class ACQUITY UPLC SM-FTN ACQUITY UPLC TUV Detector ACQUITY UPLC CM-A XEVO G2-S QTof UNIFI v1.6. Scientific Information System

LC conditions

Column: ACQUITY UPLC-BEH300 C4, 2.1x50 mm

Column temperature: 80 °C

Mobile phase A: water

Mobile phase B: acetonitrile

Mobile phase C: not used

Mobile phase D: 0.5% TFA (in water)

Detection: UV 280 nm

Total run time: 20.0 min

LC Gradient Table:

MS Conditions

Capillary: 3.0 kV

Sampling cone: 80 V

Extraction cone: 4 V

Source temperature: 125 °C

Desolvation temperature: 350 °C

Cone gas flow: 0 L/Hr

Desolvation gas flow: 800 L/Hr

Unifi Workflow

References Xie H, Chakraborty A, Ahn J, Yu Y, Dakshinamoorthy D, Gilar M, Chen W, Skilton SJ and Mazzeo J, Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies, MAbs. 2010; Jul-Aug; 2(4): 379–394.

RESULTS AND DISCUSSION Light chain (LC) and heavy chain (HC) characterization for Inflixmab

from two cell lines Infliximab (Remicade®) is a chimeric (mouse/human) IgG1 monoclonal antibody. When reduced, one IgG1 molecule dissociates into two light chains and two heavy chains.

Figure 2. The reversed-phase LC/MS chromatograms from the analysis of reduced Infliximab from two cell

lines.

Figure 3. The combined raw MS spectra and deconvoluted spectra in mirror image. The MS spectra of the

light chain (eluted around 3.5 min in Figure 2) from the SP2/0 sample and the CHO sample are displayed.

Figure 4. The combined raw MS spectra mirror image comparison between the heavy chains (eluted

around 10 min in Figure 1) for the innovator and the biosimilar

CONCLUSION With UNIFI scientific information system, Infliximab from two cell lines can be

rapidly analyzed, and the information on their structural variation can be

readily generated for the assessment of batch comparability.

The results indicate that although there are batch variations for the major

glycoforms among the innovator batches as well as the biosimilar batches,

the biggest differences were found to be between the innovator and the

biosimilar samples.

Inflixmab biosimilars have the most abundant response for the C-terminal

Lysine modified (cleaved) G0F isoform, while the innovator samples have the

most abundant response form the G0F isoform without the C-terminal

modification.

Inflixmab biosimilars have a higher response for the G0 isoform with and

without the C-terminal modification, in compare to the innovators. The

innovators have a much higher response for the G1F isoform without the C-

Terminal modification.

All of these results demonstrate that waters biopharmaceutical system

solutions with Unifi scientific system is a powerful, flexible and comprehensive

platform to provide routine analysis for biopharmaceutical industry.

Figure 5. Identified major peaks from the innovator heavy chain deconvoluted spectra are glycoforms of G0

N, G0F N, G1F N, G2F N, and Man 5 with and without the C-terminal Lysine (K) modification.

The differences between the samples can be examined based on the deconvoluted spectra in

Figure 4. It appears that infliximab from SP2/0 cells (innovator) is more heterogeneous than

that from CHO (Biosimilar). Several major glycoforms including G0 N, G0F N, G1F N, G2F N, and

Man 5 are identified with both samples, however the relative abundance for each glycoforms is

found to be very different among them. For example, the sample from CHO cells contains

higher abundance of G0 and G0F glycoforms than that of the sample from SP2/0 cell line, while

the relative percentage of G1F and G2F glycoforms in innovator sample is greater than that of

the CHO.

METHODS EXPERIMENTAL Sample Information

Three (3) batches of Remicade, infliximab purchased from Jenssen biotech,

Inc. (Horsham, PA), which were produced by SP2/0 cell line. Three (3)

batches of Infliximab produced by an alternative mammalian cell line (CHO).

Structural comparison of multiple batches of Infliximab from

two cell lines Figure 6 shows a few examples of screen shots from the report generated in

the analysis. It contains the report title page, the sample list, a component

plot which contains the relative response of the major glycoisoforms from one

of the innovator batches and biosimilar batches, a couple of distribution plots

for glycoform G0N and Man 5 MS response. It shows excellent flexibility in

meeting customized reporting requirement.

As can be seen from the component plot, the lysine-containing C-terminal

peak areas were much smaller for the innovator sample from CHO cell line as

compared to the areas observed for the same peaks in the NS2/0 cell line

biosimilar sample. This experimental result confirms that there was a lower

level of C-terminal lysine in antibodies derived from the CHO cell line. This

modification can be of importance since it is found to be sensitive to the

production process. It was determined that the relative amount of

incorporated C-terminal lysine can vary greatly between these two production

schemes.

The Unifi summary plot offers simple and direct view to demonstrate the

variation of G0 glycoform among all the samples and all the injections. The

replicate analysis for each sample generates highly reproducible

measurement, and the responses of G0 glycoform for infliximab produced by

within the each cell line are rather similar, demonstrating a great control over

the production of the antibody. On the other hand, it appears that the CHO

samples generate a much higher response for the G0N glycoform in compare

to that of the innovator batches.

Figure 6. Screen shots from the analysis report demonstrate the excellent flexibility in Unifi

report for meeting any reporting requirements.