AbstractCharacterization of the N-linked oligosaccharide (glycan) profile of a monoclonal antibody (MAb) is an important component of consistent lot release for clinical studies and marketed product support. Glycans help to define activity, efficacy and immunogenicity of MAbs. Glycan profile heterogeneity is essential for adherence to Good Manufacturing Practice principles. Capillary electrophoresis using laser-induced fluorescence (CE-LIF) has become a significant tool for performing glycan analysis of MAbs. Historically, CE-LIF methods have taken several days to perform due to the many steps required for sample preparation and analysis. In this presentation, we compare and contrast two different sample preparation techniques and three different electrophoresis methods. Materials included a PROzyme Glyko® Human IgG N-linked Glycan Library, a Beckman Coulter (BC) G20 Glucose ladder, a BC IgG Control Standard, and a Sigma Aldrich Human IgG1 Kappa. Sample preparation included deglycosylation performed using PNGase-F with either a 3-hour or an overnight 37°C incubation. Precipitation of proteins was performed using ethanol, followed by drying. Reductive amination using APTS and sodium cyanoborohydride was followed by heating for 90 minutes at 55°C. Samples were quenched, then electrophoresed using either a bare fused silica capillary or an N-CHO capillary using a BC PA 800 plus CE-LIF system. The three CE-LIF methods consisted of either 1) 240 mM borate, pH 9.0 buffer, run time 60 minutes, 2) BC carbohydrate separation (CS) buffer, run time 20 minutes, or 3) 1:1 ratio of the BC CS buffer and BC dsDNA 1000 separation gel buffer, run time 20 minutes. The overnight enzymatic deglycosylation incubation step provided no significant advantage over the 3-hour incubation. The first method resulted in acceptable resolution (Rs); however theoretical plates (N) were low. The second method had acceptable Rs and N while the third method resulted in superior N and Rs. All 3 separation methods yielded acceptable glycan profiles. The long preparation method takes 2 days. The shorter preparation method can be performed in 9 hours and when coupled with the appropriate buffer, results in a significant gain in efficiency and improved quality of results.

Materials and Methods■ Beckman Coulter Inc. PA800 plus Pharmaceutical Analysis System

equipped with a 488 nm solid state laser■ Human IgG N-Linked Glycan Library-PROzyme, Inc (p/n GKLB-005)■ Human IgG1, KAPPA – Sigma (p/n I 5154)■ PNGase Kit for deglycosylation – QA Bio (p/n E-PNG01)■ Carbohydrate Labeling Analysis Kit - Beckman Coulter, Inc (p/n 477600)

- G20 Glucose Ladder- G22 Maltose Standard- APTS- Carbohydrate assay gel (PEO)

■ dsDNA 1000 Separation gel buffer (LPA) - BC (p/n 477626)■ IgG Control Standard-BC (p/n A10663)■ Unknown MAb – Covance■ Boric Acid – Sigma (p/n B7901)

Preparation Method A(Carbohydrate Labeling and Analysis Kit from BC) (2 days)

■ Deglycosylate ~ 200 µg protein overnight at 37°C ■ Protein precipitated with chilled ethanol, supernate spiked with Maltose

marker■ Samples dried and labeled with APTS for 90 minutes at 55°C

Preparation Method B (1 day)■ Deglycosylate ~ 200 µg protein for 3 hours at 37°C ■ Protein precipitated with chilled ethanol, supernate spiked with Maltose

marker ■ Samples dried and labeled with APTS for 90 minutes at 55°C

All 3 separation methods included the following:

■ Capillary diameter: 50 µm I.D.■ Capillary length: total length(Lt) = 50.2 cm,■ length to detector (Ld) = 40.2 cm

Method parameters were as shown above unless otherwise indicated.

Characterization of Recombinant Monoclonal Antibody N-Linked Oligosaccharides Using CE-LIFJoan Garrison, Kim Wisman, Peter Holper, Debbie Drenzyk and Renee’ RickeCovance Laboratories Inc., Greenfield, Indiana

Separation Parameters Method 1 (SPM1)■ Bare Fused Capillary■ 240 mM Borate Buffer, pH 9.0■ 60 minute run time■ Capillary temperature: 18°C■ Sample storage temperature: 20°CSeparation Parameters Method 2 (SPM2)■ Neutral Capillary■ PEO Gel■ 20 minute run time■ Capillary temperature: 20°C■ Sample storage temperature: 10°CSeparation Parameters Method 3 (SPM3)■ Neutral Capillary■ 1:1 mixture of PEO and LPA Gels■ 20 minute run time■ Capillary temperature: 20°C■ Sample storage temperature: 10°C

Note: Initial results using SPM1 yielded acceptable Rs, but low N values were observed. After a number of analyses, the SPM1 results were irreproducible, and require additional investigation. Therefore, at this time the SPM1 results were not reported.

Peak identifications are: 1- G0F, 2- G1F, 2’- G1’F, 3- G0FB, 4- G2F,

5- G2FS1. (see Figure 3 for electropherograms)

Figure 1. Deglycosylation and APTS labeling of glycans. 1. Glycan cleavage of an N-linked oligosaccharide using PNGase F. 2. APTS labeling of a complex oligosaccharide.

1.

2.

Table 1. Glycan Abbreviations and Descriptions

Abbreviation Description Compound Name ~ MW (Da)

G0 Asialo, agalacto, biantennary complex NGA2 1317

G0F Asialo, agalacto, biantennary complex, core substituted with fucose NGA2F 1463

G1F/G1’F Asialo, mono-galactosylated, biantennary complex, core substituted with fucose NA2G1F 1626

G0FBAsialo-, agalacto-, biantennary core, core- substituted with fucose and

bisecting N-acetylglucosamineNGA2FB 1667

G2F Asialo, galactosylated, biantennary complex, core- substituted with fucose NA2F 1787

G2S1FMono-sialyate, galactosylated,

biantennary complex, core- substituted with fucose

A1F 2079

Figure 2. Glucose Ladder. The number designation for the glucose ladder standard refers to the number of glucose subunits making up the standard. Separation conditions were 30kV following a 1.5 psi injection for 20 seconds using SPM2. Insert: Glucose ladder separation using SPM1. Separation conditions were 22kV following a 1.5 psi injection for 20 seconds (Note: Glucose ladder preparation contained in Beckman Coulter Carbohydrate kit.)

Figure 3. Glyko® Human Glycan Library. Black – Analysis performed using SPM3. Blue – Analysis performed using SPM2. Peak identifications are: 1- G0F, 2- G1F, 2’- G1’F 3- G0FB, 4- G2F, 5- G2FS1. Separation conditions were 30kV following a 1.5 psi injection for 20 seconds Insert: Glycan separation using SPM1. Separation conditions were 22kV following a 1.5 psi injection for 20 seconds.

Figure 4. Beckman Coulter IgG control method preparation comparison. Method A (black) and Method B (blue) using SPM2 (top figure). Method A (black) and Method B (blue) using SPM3 (bottom figure). Separation conditions were 20 kV following a 1.5 psi injection for 20 seconds. Capillary 60.2 cm(Lt) and 50.2 cm (Ld). Insert: Method A using SPM1. Separation conditions were 22kV following a 1.5 psi injection for 20 seconds.

1

10 5

15

20

25

30

RF

U

Maltose marker

Maltose marker

G0F

G0F

G1F

G1F

G1’F

G1’F

G2F

RF

U

RF

U

Maltose marker

Maltose marker

G0F

G0F G1F

G1F G1’F

G1’F

G2F

1

2

2’ 3

3

2’

2

1

4

4

5

5

RF

U

Figure 5. Sigma IgG1, Kappa method preparation comparison. Method A (black) and Method B (blue) using SPM2. Separation conditions were 30kV following a 1.5 psi injection for 20 seconds. Insert: Method A (black) and Method B (blue) using SPM1. Separation conditions were 22kV following a 1.5 psi injection for 20 seconds.

maltose

G0

G0F G1F G1’F

G2F

RF

U

Figure 6. MAb (Covance) method preparation comparison. Method A (black) and Method B (blue) using SPM3. Separation conditions were 30kV following a 1.5 psi injection for 20 seconds. Insert: Method A (black) and Method B (blue) using SPM1. Separation conditions were 22kV following a 1.5 psi injection for 20 seconds.

maltose

G0

G0F

G1F

G1’F

RF

U

Maltose marker

G0

G0F G1F

G1’F

RF

U

Results and DiscussionPreparation Methods■ Method B (1-day prep) provides a considerable gain in efficiency and

yields similar results to Method A (2-day prep).

Separation Methods■ SPM1 (Borate buffer) demonstrated acceptable resolution, but was less

efficient (run time = 60 minutes).

■ SPM2 (PEO) also showed acceptable resolution

■ SPM3 (1:1 PEO /LPA) illustrated superior resolution of glycans, but with suppressed signal (RFUs).

Conclusion■ Data has shown that capillary electrophoresis is capable of separating

N-linked oligosaccharides using the 3 separation methods indicated above.- SPM2 and SPM3 provided considerable advantages, such as better reproducibility and shorter run time, than SPM1. - Method B (1-day prep), when coupled with the appropriate separation method, results in a significant gain in efficiency.- Further optimization is required to identify additional complex oligosaccharides and to investigate alternative separation medias.

AcknowledgementsWe would like to thank Andre´ Johnson, James Bourdage, Ray Kaiser and Monica Whitmire for their support and technical guidance. Additionally, we would like to thank Beckman Coulter, Inc. for supplying many of the materials used to perform the analyses.

ReferencesRampal, S., Gennaro, L., Lies, M. Beckman Coulter, IB-15285A, (2011).

Knudson, V.F., Simonian, M.H. Beckman Coulter, Application Information, A-1986A, (2004).

Carbohydrate Labeling and Analysis Application Guide, Beckman Coulter, Inc, (2011).

Chen, F.-T. A., Dobashi, T.S., Evangelista, R.A. Glycobiology, Vol. 8, no 11, 1045-1052, (1998).

Human IgG N-Linked Glycan Library – Prozyme, Inc. – Certificate of Analysis insert.

PNGase F Specifications-Protocols – QA Bio, E-PNG01.

Beckman Coulter – Brea, CA. www.beckmancoulter.com or www.celeader.com

Table 2. Resolution (Rs) for Glyko® HumanGlycan Library

Resolution (Rs)

Peak label SPM3 SPM2

1 37.9 18.2

2 6.9 6.2

2’ 2.0 1.8

3 8.1 6.1

4 5.0 5.1

Table 3. Theoretical Plate Count (N) for Glyko® Human Glycan Library

Theoretical plate count (N)

Peak label SPM3 SPM2

1 290824.6 187346.1

2 261610.5 198002.8

2’ 112039.0 222714.4

3 232385.7 141552.5

4 217265.9 218066.2

5 258277.4 75063.1

Note: Initial results using SPM1 yielded acceptable Rs, but low N values were observed. After a number of analyses, the SPM1 results were irreproducible, and require additional investigation. Therefore, at this time the SPM1 results were not reported.

Peak identifications are: 1- G0F, 2- G1F, 2’- G1’F, 3- G0FB, 4- G2F,

5- G2FS1. (see Figure 3 for electropherograms)

Note: This table indicates the compound name, abbreviations, and molecular weights for each glycan species.

Presented at the2012 AAPS Annual Meeting and ExpositionChicago, Illinois14–18 October 2012

Characterization of Recombinant Monoclonal Antibody N-LinkedOligosaccharides Using CE-LIF

Joan Garrison, Kim Wisman, Peter Holper, Debbie Drenzyk and Renee’ RickeCovance Laboratories Inc., Greenfield, Indiana

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Web Site: www.covance.com

Covance is an independent, publicly held company with headquarters in Princeton, New Jersey, USA. Covance is an independent, publicly held company with headquarters in Princeton, New Jersey, USA. Covance is the marketing name for Covance Inc. and its subsidiaries around the world. Covance is the marketing name for Covance Inc. and its subsidiaries around the world.

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