Optimizing the Quality and Accuracy of Biopharmaceutical ... · 1100/1200 or Waters 2690/2695/2796 HPLC systems. Potential issues of salt-based IEC methods Slide 10 2/12/2013 run

Optimizing the Quality and Accuracy of

Biopharmaceutical Analysis and Characterization

Broadcast Date: Tuesday, February 19, 2013

Time: 11AM EST, 8AM PST

Sponsored by





Your Moderator

Tamlyn Oliver Managing Editor

Genetic Engineering & Biotechnology News



Galahad Deperalta Scientist

Protein Analytical Chemistry

Genentech

Ion Exchange Chromatography (IEC) of

Therapeutic Antibodies (mAbs):

Experiences Working With a

Bio-inert/Bio-compatible HPLC

Galahad Deperalta

Scientist, Protein Analytical Chemistry

Genentech, A Member of the Roche Group

GEN Webinar

February 19, 2013

Slide 5 Presentation Outline

• Brief introduction on therapeutic antibodies (mAb) and heterogeneity

• Brief overview on Ion-exchange Chromatography (IEC) as tool to monitor

charge heterogeneity

• Working with a Bio-Inert HPLC system for IEC analysis: Case Study mAb #1

• Working with a Bio-Inert HPLC system for IEC analysis: Case Study mAb #2

• Summary

• Acknowledgements

Slide 6 2/12/2013 Therapeutic monoclonal antibodies (mAbs)

• Recombinant monoclonal antibodies (mAbs) have become widely accepted therapies for

various forms of cancer and immunologic diseases.

• Post-translational modifications (PTMs), introduced at various points of the manufacturing

process or during storage, add heterogeneity to the mAb.

• PTMs such as glycosylation, deamidation, oxidation, isomerization, aggregation and other

product-related variants may affect the mAb’s efficacy, stability, and safety profile. To better

understand and control the levels of these variants, it’s important to monitor and

characterize them during the course of development and manufacturing.

• PTM’s can affect the surface charge of the mAb. Thus, such product-related variants can

be monitored by methods (such as IEC) that resolve on the basis of surface charge

differentiation.

• Elucidating the charge heterogeneity of a therapeutic mAb is an important aspect of

determining its purity.

Slide 6

Slide 7 2/12/2013 IgG structure and common charge variants Slide 7

Racacniello, V., “Antibody .“ Diagram . Virology Blog 22 Jul 2009

<http://www.virology.ws/2009/07/22/adaptive-immune-defenses-

antibodies/>

Example of modifications that typically

elute as acidic variants

Examples of modifications that typically

elute as basic variants

Deamidation, Glycation,

Glycosylation, High mannose,

Sialylation, Fragments, Cysteinylation,

Carbamylation

Oxidation, Isomerization, Succinimide,

Amidation, Aglycosylation, Fragments,

Aggregates, Incomplete removal of

leader sequence, C-terminal lysine

Slide 8 2/12/2013 Monitoring mAb charge variants by Ion-Exchange Chromatography (IEC)

Slide 8

• EMA: Guideline on Development, Production, Characterization and Specifications for

Monoclonal Antibodies and Related Products

Section 4.3.4: “In addition, suitable methods should be proposed to qualitatively and

quantitatively analyse heterogeneity related to charge variants.”

Section 4.4.2: “…separation methods based on charge heterogeneity should be considered to

quantitatively and qualitatively monitor charge variants.”

• Analytical methods that can be employed to elucidate charge-related heterogeneity in

therapeutic mAbs include isoelectric focusing (IEF), imaged capillary isoelectric focusing

(icIEF), and ion-exchange chromatography (IEC).

• Benefits of IEC: Relatively simple to enrich/isolate charge variants for further, extended

characterization studies.

• Two types of IEC can be used: Cation-exchange chromatography (CEX) or Anion-exchange

chromatography (AEX). Focus of this presentation will be on CEX.

Slide 9 2/12/2013 Monitoring mAb charge variants by IEC: Focus on Cation-Exchange Chromatography (CEX)

Slide 9

• CEX widely used since many therapeutic mAbs have a basic isoelectric point. mAbs are

retained on CEX by interaction of their His, Lys, and Arg residues with the negatively

charged resin of the column. mAbs can be eluted off the column by increasing the salt

concentration (salt-based gradient) or the pH (pH gradient) of the mobile phase.

• In CEX, acidic species/variants elute earlier than the main peak, basic species/variants

elute after the main peak.

• Column commonly used for CEX analysis of therapeutic mAbs: Dionex ProPac WCX-10

(WCX: weak cation exchanger)

• ProPac WCX-10 column “has become the gold standard for antibody analysis.” (Vlasak and

Ionescu, 2008)

• Mobile phase, mobile phase pH, salt concentration and gradient, column temperature need

to be optimized for each therapeutic mAb.

• Traditionally, we have run CEX methods, using the Dionex WCX column, on Agilent

1100/1200 or Waters 2690/2695/2796 HPLC systems.

Slide 10 2/12/2013 Potential issues of salt-based IEC methods run on stainless steel HPLC systems

• Some risks involved with using traditional stainless steel-based HPLC systems.

• Long-term use of high salt mobile phases can lead to corrosion of metal components.

• Metal complexes can form from corroded components and leach onto the IEC column,

ultimately leading to a degradation of column performance (e.g loss of resolution, baseline

instability, etc.).

• Chelation of the mAb by the metals leached onto the column can lead to secondary

interactions and subsequent peak tailing. Contact of the mAb with corroded surfaces in the

flow path can also lead to chelation.

• Adsorption of analytes/proteins onto corroded or rusted surfaces is also possible.

• Corrosion of metal components can also ultimately damage the HPLC instrument itself.

• System contamination by microorganisms is also a concern when constantly running high salt

mobile phases.

Slide 10

Slide 11 2/12/2013 Assessment of Bio-Inert HPLC systems

• Collaboration with Agilent began in 2008 to evaluate a bio-inert HPLC system for improved

biotherapeutic applications. Goal was to evaluate the benefits of an HPLC system with

corrosion-proof components for salt-based chromatography methods.

• From 2008-2009, a prototype 1200 BioLC system with an all titanium flow path was assessed

at Genentech for IEC applications.

• Positive results observed in terms of maintenance of baseline stability and prolonging IEC

column lifetimes. Several therapeutic mAbs were tested on the system.

Slide 11

2/12/2013 Assessment of Bio-Inert HPLC systems

• In 2010, collaboration with Agilent extended to assessing a pre-production model of the 1260

Infinity Bio-Inert Quaternary HPLC.

• General strategy for assessment: Challenge the system by running long sequences of salt-

based gradient IEC/CEX methods. Baselines, peak resolution/profiles, peak quantitation

monitored over the course of the sequence.

• mAb IEC methods that were known to be problematic (unstable baselines, subsequent

inconsistent profiles and quantitation) on non-Bio-inert HPLC’s were targeted for assessment

on the Agilent 1260.

• Case studies from two mAb’s weak cation-exchange (WCX) chromatography methods will be

presented here.

Slide 12

• 100% bio-inert

• Samples do not touch metal surfaces

• No stainless steel in the mobile phase flow path

• New capillary technology (PEEK tubing encased

in stainless steel)

• High corrosion resistance

• Titanium-based pump

• Metal-free detector flow cell

Slide 13

2/12/2013

Case Study mAb #1 IEC analysis

• IgG1 therapeutic mAb

• Column: Dionex ProPac WCX-10 4x250mm

• Mobile Phase A: MES buffer, Mobile Phase B: NaCl in A (linear gradient)

Representative Agilent 1100 chromatogram

mAb #1 IEC profile problems on SS-based HPLC:

Repeat sample injection #1 vs #37 in a long sequence

Anomalous peaks grow as

sequence progresses

Slide 14

Acidic region Basic region

Main Peak

Red trace: Sample injection #1

Blue trace: Sample injection #37

mAb #1 IEC problems on SS-based HPLC:

baseline instability Slide 15

• Baseline instability causes problems with consistent integration

• Higher risk of failing system suitability quantitative criteria

Acidic region

Basic region

Main Peak

mAb #1 IEC problems on SS-based HPLC: Example of a test session failure

Acidic:25.7 %

Main: 65.4%

Basic: 8.9%

Acidic:21.8 %

Main: 65.2%

Basic: 13.0%

Reference:

DR5904-1

Slide 16

N= 31 independent QC test sessions

Number of times both bracketing

Reference Material injections within

the same sequence did not pass

quantitative system suitability

criteria: 5 out of 31 test sessions

Failure rate : (~16 %)

First bracketing Reference Material

Second bracketing Reference Material

Sample Injection # 1

Sample Injection # 21

Slide 17 Mab #1 IEC on prototype Agilent 1200 BioLC: Example of a long sample sequence run

• IEC baseline and profiles appear

more consistent over the course of

the sequence.

• Anomalous peaks not observed to

grow over the course of the

sequence.

Mab #1 IEC on the Agilent Bio-Inert 1260: Example of a very long sample sequence run

mA

U

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 62.5 65.0 67.5 70.0 -50

0

50

100

150

200

250

300

350

400

450

500

min

Run #44

Run #35

Run #24

Run #13

Run #2

Blank #1

Mobile Phase A: MES buffer

Mobile Phase B: NaCl in A

Column: Dionex ProPac WCX-10 4 x 250 mm

mA

U

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 62.5 65.0 67.5 70.0 -50

0

50

100

150

200

250

300

350

400

min

Blank #12

Blank #23

Blank #34

Blank #45

Run #44

Slide 18

Formulation blank injections

throughout the sequence

Aaron Wecksler

Acidic Region

Main Peak

Basic Region

0.1 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 26.3 27.5 28.8 30.0 31.3 32.5 33.8 35.1 -49

-25

0

25

50

75

100

125

150

175

200

225

250

Acidic Region

Main Peak

Basic Region

Formulation Blank

Mab #1 IEC Trace

mA

U

min

% Acid

Region % Main Peak

% Basic

Region

Average ± STD

(%RSD)

N=40

19.8 ± 0.4

(2.1%)

76.7 ± 0.5

(0.6%)

3.5 ± 0.2

(5.9%)

Slide 19 mAb #1 IEC on the Agilent Bio-Inert 1260

Comparison of mAb #1 IEC runs on the Agilent Bio-Inert 1260 to

previous/historical IEC runs on non-Bio-Inert HPLCs:

• Stable baselines and consistent profiles maintained over long sequence runs.

• Anomalous peaks not associated with the protein are not observed.

• Consistent quantitative data, system suitability criteria consistently passed.

• Useable column lifetimes appear to be extended.

Aaron Wecksler

Slide 20 Case Study mAb #2 IEC analysis

• IgG1 therapeutic mAb

• Column: Dionex ProPac WCX-10 4x250mm

• Mobile Phase A: ACES buffer , Mobile Phase B: NaCl in A (linear gradient)

Representative Waters 2690/2695 chromatogram

Example of an acceptable baseline for mAb #2 IEC

(Formulation buffer blank injection)

Normal baseline, no unusual or drastic humps

within the expected protein elution region

Slide 21

Jeannie Kwong and Armando Cordoba

Chromatogram from a SS-based HPLC

Example of an acceptable profile and integration for mAb #2 IEC


• Acceptable (“normal”) baseline and product profile

• Meets quantitative system suitability criteria

Slide 22

Acidic region

Basic region

Main Peak


Example of poor or unacceptable baseline for mAb #2 IEC

(Formulation buffer blank injection)

Pronounced Baseline hump affects integration

Slide 23


This peak increases

over the course of a

sequence

This peak increases over the

course of a sequence


Example of unacceptable profile and integration for mAb #2 IEC

This peak increases

over the course of a

sequence

This peak increases over the

course of a sequence

Sample does not pass system suitability

(integration affected by baseline hump)

Slide 24



0.2 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0 62.8

-1.9

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

26.0

29.2

1 - AVEGFIEC060311JK_LAURENF20 #5 [modified by kwongj1] Avastin ref mat (408-3) UV_VIS_1

2 - AVEGFIEC060311JK_LAURENF20 #14 Avastin ref mat (408-3) UV_VIS_1



5 - AVEGFIEC060311JK_LAURENF20 #29 Form buffer blank UV_VIS_1mAU

min

5

4

3

2

1

WVL:280 nm

Example of mAb #2 IEC long sequence with poor baseline and profiles (run on a SS-based HPLC)

Formulation blank, injection #29

Ref mat injection #21




Slide 25


• IEC profiles become progressively worse

• Quantitative system suitability criteria not met

Slide 26

Time consuming passivation or cleaning procedures instituted between or within

IEC sequence runs of mAb #2 run on non-Bio-inert HPLC’s (procedure provided by

Agilent):

1) 30% Phosphoric Acid at 3ml/min for 20 min. (and/or 6N nitric acid 60ml)

2) 0.5M NaOH at 1ml/min for 4 hours

3) MilliQ H2O at 5ml/min for 4 hours

4) 40mM EDTA at 5ml/min for 3 hours

5) MilliQ H2O at 5ml/min for 4 hours

Also tried adding a wash step by running 0.5N NaOH for ~5 minutes through the

column at the end of each injection. Baseline integrity would temporarily be

restored, but it was observed that some basic peaks would sometimes be lost.

The passivation and cleaning procedure were only temporary solutions, as the

baselines would eventually become unacceptable again as IEC sequences of Mab

#2 continued to be run on these non-Bio-inert HPLC’s. Baseline problems were

reproducible using different Dionex WCX-10 columns and lots, and on different

HPLC instruments.

Jeannie Kwong

Passivation and cleaning procedures attempted in order to restore baseline integrity for mAb # 2 IEC

Example of mAb #2 IEC sequence run on the Agilent Bio-Inert 1260

1.7 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0 62.5

-2.5

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

25.0

1 - AvastinIEC062612JK_Lauren18 / Form buffer blank / #22 DAD1_A__Sig_280_8_Ref_360_100

2 - AvastinIEC062612JK_Lauren18 / Avastin Ref Mat 408-3 / #1 [modified by kwongj1] DAD1_A__Sig_280_8_Ref_360_100

3 - AvastinIEC062612JK_Lauren18 / Avastin Ref Mat 408-3 / #10 [modified by kwongj1] DAD1_A__Sig_280_8_Ref_360_100

4 - AvastinIEC062612JK_Lauren18 / Avastin Ref Mat 408-3 / #21 [modified by kwongj1] DAD1_A__Sig_280_8_Ref_360_100mAU

min

4

3

2

1

Formulation blank




Slide 27


• IEC baseline and profiles maintain

consistency over the course of a long

sequence.

• No anomalous baseline humps observed.

• Quantitative system suitability criteria met for

all bracketing Reference Material samples.

Acidic Region

Basic Region

Main Peak

mAb #2 IEC sequence on the Agilent Bio-Inert 1260: Analyst 1 using Dionex column 1

-0.1 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.1 -40

-20

0

20

40

60

80

100

120

140

160

180

196

mA

U

min

Acidic Region

Main Peak

Basic Region

Ref Mat injection #14


Formulation Blank

Slide 28

Aaron Wecksler

• IEC baseline and profiles maintained consistency over the course of a long sequence.

• Quantitative system suitability criteria met for bracketing Reference Material samples.

% Acid

Region

% Main

Peak

% Basic

Region

Ref Mat

%RSD

N=5

25.9 ± 1.2

(4.6%)

69.4 ± 0.6

(0.9%)

4.1 ± 0.3

(6.1%)

-0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 49.8 -47

-25

0

25

50

75

100

125

150

175

200

225

250

mA

U

min

Ref Mat Run #3

Ref Mat Run #2

Formulation Buffer

Ref Mat Run #10

Slide 29 mAb #2 IEC sequence on the Agilent Bio-Inert 1260: Analyst 2 using Dionex column 2

• Stable baselines and consistent profiles maintained over long sequence runs.

• No anomalous peaks in the baseline are observed.

• Consistent quantitative data, system suitability criteria consistently passed.

Jeannie Kwong

Acidic Region

Main Peak

Basic Region

mAb #2 pH gradient IEC test sequence run on the Agilent Bio-Inert 1260

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 -20

0

20

40

60

80

100

120

140

160

180

200

220

min

mA

U




Formulation Blank

Mobile Phase A: Tris/Imidazole/Piperazine, pH 6.0

Mobile Phase B: Tris/Imidazole/Piperazine, pH 9.5

Column: Dionex ProPac WCX-10 4 x 250 mm

% Acid

Region % Main Peak

% Basic

Region

Ref Mat

N=3

24.2 ± 1.5

(6.3%)

63.4 ± 2.6

(4.0%)

12.6 ± 1.0

(8.0%)

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 -30

0

20

40

60

80

100

120

140

160

mA

U

min

Acidic Region

Main Peak

Basic Region

Mab #2 pH Gradient Trace

Formulation Blank

Slide 30

• pH gradient IEC baseline and profiles

maintained consistency over the course of a

long sequence.

Slide 31 2/12/2013 Summary

• Collaboration with Agilent over the past few years has led to assessments of Bio-inert HPLC systems for

the chromatographic charge variant analysis of therapeutic mAbs.

• Two mAbs, with salt-based IEC/CEX methods that utilize Dionex ProPac WCX-10 columns, that have

shown to be problematic on stainless steel HPLC systems were evaluated on the Agilent Bio-Inert 1260.

The issues previously observed with these methods included baseline instability (anomalous humps or

peaks, baseline drifts or shifts), inconsistent resolution, and inconsistent quantitation that result in failed

system suitability criteria.

• Positive results were observed in both case study mAbs’ IEC methods on the Agilent Bio-Inert 1260.

• Over the course of long IEC sequence runs on the Bio-Inert system, baseline integrity was maintained (as

monitored by formulation blank injections). The chromatographic profiles and resolution remained

consistent, and quantitative system suitability criteria were consistently met.

• No cleaning or passivation methods were needed to maintain the positive results.

• Data from multiple sequence runs of mAb #1 and mAb #2 suggest that useful column life may be extended

with the use of a Bio-Inert system.

• Future assessments on the Agilent Bio-Inert 1260 : UHPLC IEC and SEC method evaluation, additional pH

gradient IEC method runs (wish list: in-line pH or conductivity monitor, and higher pressure capability), AEX

method evaluations, HIC method evaluations, RP-HPLC applications (is the system sufficiently robust to

switch between aqueous/salt-based and organic mobile phases)

Slide 31

Slide 32 Acknowledgements

Genentech

Armando Cordoba

Jeannie Kwong

Lisa Vampola

Frank Macchi

Milady Ninoneuvo

Yun Lou

Connie Lu

Tim Spirakes

Taylor Zhang

Hadil Suliman

Melissa Alvarez

Minako Pazdera

Dell Farnan

Victor Ling

John Stults

Reed Harris

Lancaster Labs

Aaron Wecksler

Agilent

Michael Yap

Taegen Clary

Stefan Jenkins

Lisa Zang

Stefan Falk-Jordan

Martin Vollmer

Emma Liu

GEN

Tamlyn Oliver

Some selected reading for more in-depth information on mAb charge variants and IEC

Harris, R.J., Kabakoff, B., Macchi, F.D., Shen, F.J., Kwong, M., Andya, J.D., Shire, S.J., Bjork, N., Totpai, K.,

& Chen, A.B. Identification of multiple sources of charge heterogeneity in a recombinant antibody. Journal of

Chromatography B 2001; 752:233-245

Rea, J., Wang, Y., Moreno, T., Parikh, P., Lou, Y., Farnan, D. Monoclonal antibody development and

physicochemical characterization by high performance ion exchange chromatography. Innovations in

Biotechnology, Dr. Eddy C. Agbo (Ed.) 2012

Khawli, L., Goswami , S., Hutchinson, R., Kwong, ZW., Yang, J., Wang, X., et al. Charge variants in IgG1:

Isolation, characterization, in vitro binding properties and pharmacokinetics in rats. mAbs.2010;2:613–624

Du, Y., Walsh, A., Ehrick, R., Xu, W., May, K., Liu, H. Chromatographic analysis of the acidic and basic

species of recombinant monoclonal antibodies. mAbs 2012; 4:578-585

Vlasak, J., & Ionescu, R. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods.

Current Pharmaceutical Biotechnology 2008; 9: 468–481

Rao, S., & Pohl, C. Reversible interference of Fe3+ with monoclonal antibody analysis in cation exchange

columns. Analytical Biochemistry 2011; 409:293-295,

Farnan, D., Moreno, T. Multiproduct high-resolution monoclonal antibody charge variant separations by pH

gradient ion-exchange chromatography. Anal. Chem 2009; 81: 8846–8857

Slide 33



Mike Kimzey, Ph.D. Senior Scientist

ProZyme

High-throughput Screening for N-Glycosylation

Mike Kimzey February 19th 2013 GEN webinar

Outline

• Optimized N-glycan Sample Preparation

• High-throughput Methods

• Innovator N-glycan Target Range

• Cell-Culture Optimization for N-glycans

36

GlykoPrep™ Overview

Purify target

Denature & immobilize

Digest with N-Glycanase®

Fluorescent label

Cleanup & elute in water

Crude sample

Purified glycoprotein

Analysis • Modular design

• Quantitative sample preparation 2 - 5 hours

– Integrated purification (e.g., MAbs)

– Rapid, unbiased N-glycan release

– Labeling: InstantDye™ or Rapid-Reductive-Amination™

• Manual or automated operation

37

AssayMAP® Cartridges

38

Label Choices

Glycoprotein N-Glycanase®

H2O

NH3

2-AB

InstantDye™

APTS

Instant AB

Rapid-Reductive-Amination™

39

++clearance

Terminal Sialic Acid -clearance

ADCC

ADCC: Antibody-Dependent Cell-mediated Cytotoxicity CDC: Compliment-Dependent Cytotoxicity

CDC

Therapeutic Effects of N-Glycans

40

+clearance

Overview of Cell-line Selection/Cell-culture Optimization

Early Screening Emphasizes capacity and speed over accuracy

6 Months

Later screening Increases in complexity and emphasizes fit to manufacturing process

Target Binding Aggregation N-Glycan

Charge Heterogeneity

Growth (Titer)

Fc Functional Assays

500 – 1000 clonal

cell lines 100 – 200 10 – 20 production cell line

41 Courtesy of Mark Melville and Joe Siemiatkoski of Epirus Biopharmaceuticals , Inc.

Characterization of Peaks (MabThera B6069) R

FU

Minutes

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

No exoglycosidase

+ Sialidase A

+ Sialidase A + β(1-4)-Galactosidase

+ Sialidase A + β(1-4)-Galactosidase + β-N-acetylhexoseaminidase

+ Sialidase A + β(1-4)-Galactosidase + β-N-acetylhexoseaminidase + α-Mannosidases

42

N-Glycan Profile of Herceptin

010203040506070

0 2 4 6 8 10

Minutes

% B

uff

er B

wash 43

Defining Target Range: Rituximab and Bevacizumab Lots

Drug Name Trade Name Vendor Lot # Vendor Expiry Vendor

Rituximab MabThera B6055 Aug-13 Roche

Rituximab MabThera H151B01 Oct-13 Roche

Rituximab MabThera B6069 Nov-13 Roche

Rituximab Rituxan 919862 Jun-13 Genentech

Rituximab Rituxan 930812 Sep-13 Genentech

Rituximab Rituxan 930814 Oct-13 Genentech

Rituximab Rituxan 938802 Nov-13 Genentech

Rituximab Rituxan 944794 Jan-14 Genentech

Bevacizumab Avastin H0007 B08 Mar-12 Roche

Bevacizumab Avastin H0106 B02 Dec-12 Roche

Bevacizumab Avastin H0107B01 Jan-13 Roche

Bevacizumab Avastin H0109B01 Mar-2013 Roche

Bevacizumab Avastin H0007B23 Mar-2013 Roche

Bevacizumab Avastin H0100B03 Oct-11 Roche

Bevacizumab Avastin 954131 Feb-12 Genentech

Bevacizumab Avastin 932315 Aug-12 Genentech

Bevacizumab Avastin 919257 Oct-12 Genentech

Bevacizumab Avastin 927070 Nov-12 Genentech

Bevacizumab Avastin 469053 Apr-13 Genentech

Bevacizumab Avastin 960470 Jul-13 Genentech

Bevacizumab Avastin 971630 Oct 2013 Genentech

Bevacizumab Avastin 469053 Apr-2014 Genentech

44

Table of Replicates--Rituximab

Day

Trade Name lot 1 2 3 4 total

MabThera H151B01 10 1 4 15

MabThera B6069 2 2 4

MabThera B6055 2 2 4

Rituxan 919862 2 4 3 7 16

Rituxan 930814 2 3 2 8 15

Rituxan 938802 2 4 2 8 16

Rituxan 930812 2 4 3 9

Rituxan 944794 2 3 5 4 14

45

Table of Replicates-- Bevacizumab

Day lot 1 2 3 total

469053 6 6 919257 4 4 927070 4 4 927070 4 4 932315 4 4 954131 4 4 960470 2 2 971630 5 4 9

H0007B08 4 4 H0007B23 5 4 9 H0100B03 2 2 H0106B02 4 4 H0107B01 2 2 H0109B01 4 4

46

0

5

10

15

20

25

30

35

40

45

MabThera H151B01

MabThera B6069

MabThera B6055

Rituxan 944794

Rituxan 919862

Rituxan 930814

Rituxan 938802

Rituxan 930812

Rituximab % Peak Area Comparison by Lot

47

Bevacizumab % Peak Area Comparison by Lot

0

10

20

30

40

50

60

70

80

90

G0-N G0F-N G0 G0F Man5 G1F[6] G1F[3] G2F

469053

919257

927070

932315

954131

960470

971630

H0007B08

H0007B23

H0100B03

H0106B02

H0107B01

H0108B01

H0109B01

48

Cell-Culture Optimization

• Two rounds of cell-culture optimization during a time course

• Round 1: Media

• Round 2: Additives

49

Round 1: Media

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

0 2 4 6 8 10 12

Media A

Media A

Media A, Mod 1

Media A, Mod 2

Media A, Mod 3

Media A, Mod 4

Media B

Days in Shake Flasks

Target Range

a b c d e f g h

% N

-Gly

can

1

50

Round 2: Additives

0 1 2 3 4 5 6

P23307SF-9

P23307SF-10

P23307SF-11

P23307SF-12

P23307SF-13

P23307SF-14

P23307SF-15

P23307SF-16

% N

-Gly

can

1

Days in Shake Flasks

Target Range

Round 1 Media Control

a

b

c d

e f g

h

51

Agilent AssayMAP Bravo

52

Comparison of %CV, Peaks >2%

Bravo Spin

average 0.47% 1.32%

min 0.27% 0.02%

max 1.43% 5.33%

median 0.38% 0.92%

53

0

5

10

15

20

25

30

35

40

45

A C E G A C E G A C E G A C E G A C E G A C E G A C E G A C E G A C E G A C E G A C E G A C E G

1 2 3 4 5 6 7 8 9 10 11 12

A1F

A2F

G0

G0F

G0F-N

G1

G1F[3]

G1F[6]

G2

G2F

Man 5

Man 6

Run Chart for Relative % Area

54

Column

Row

Innovator Lot #1

Innovator Lot #2

Innovator Lot #3

Innovator Lot #4

Conclusions

• GlykoPrep provides fast, high-throughput, high-quality analysis of the lot-to-lot variability of innovator molecule N-glycan profiles.

55

Conclusions

GlykoPrep provides fast, high-throughput, high-quality analysis of the lot-to-lot variability of innovator molecule N-glycan profiles.

• Glycoproteins to labeled N-glycans in < 4 hours

56

Conclusions

GlykoPrep provides fast, high-throughput, high-quality analysis of the lot-to-lot variability of innovator molecule N-glycan profiles.

Glycoproteins to labeled N-glycans in < 4 hours

• Evaluation of a variability of the N-glycan profiles of a number of lots ensures a wider target for biosimilar development. An assessment of the lot-to-lot variability of the innovator molecule is the first step in biosimilar product development.

57

Conclusions- continued

• Screening for N-glycan profiles, along with titer early in the process, ensures the selection of strains capable of delivering targeted N-glycan profiles.

58

Conclusions- continued

Screening for N-glycan profiles, along with titer early in the process, ensures the selection of strains capable of delivering targeted N-glycan profiles.

• The N-glycan profile can change rapidly over the cell-culture time course, suggesting the utility of bioreactor monitoring with GlykoPrep to better choose time of harvest to match the N-glycan target range of the innovator molecule.

59

Acknowledgements

ProZyme Ted Haxo Jo Wegstein Sybil Lockhart Jennie Truong Shiva Pourkevah Vicki Woolworth Susan Fuller Zoltan Szabo Justin Hyche

60

Agilent Steve Murphy Zach Van Den Heuvel Michael Bovee Adam Krahenbuhl Jennifer Reich Scott Fulton Rachel Bolger Randy Bolger

High-throughput Screening for N-Glycosylation



Optimizing the Quality and Accuracy of Biopharmaceutical

Analysis and Characterization

Q&A



Thank You For Attending



Broadcast Date: Tuesday, February 19, 2013

Time: 11AM EST, 8AM PST

Sponsored by

Documents

Optimizing the Quality and Accuracy of Biopharmaceutical ... · 1100/1200 or Waters 2690/2695/2796 HPLC systems. Potential issues of salt-based IEC methods Slide 10 2/12/2013 run