Your Biology Resource - Sigma-Aldrich · PDF fileYour Biology Resource Volume 8 ... PAPELLG GP FabRICATOR 119 120 PAPPVA GP FabRICATOR ... MAbs, 2, 500–507 (2010). 2. Beck, A. et

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Volume 8 | No. 2

New Enzymes for Antibody Characterizationfrom Sigma-AldrichRobert Gates Market Segment Manger, Sigma Life Science

Antibodies now make up the fastest growing category of therapeutic drugs. Even with today’s highly-defined recombinant humanized antibodies, the control and analysis of micro-heterogeneities relating to functional impact have become critical to the quality by design paradigm.1 Alain Beck and his colleagues at the Centre d’Immunologe at Pierre Fabre are leading the efforts to identify and characterize the criteria for therapeutic antibody analysis and quality control.2,3

Among the areas of antibody peptide and glycan analysis currently used or under investigation:

• Glycoform variability mainly relating to heterogeneous N-linked glycan populations

• Charge variants resulting from several types of protein modifications

• Amino acid modifications such as Asparagine deamidation, Aspartate isomerization, and formation of N-terminal pyroglutamate

• Cysteine-related variants and oxidation states

• Positive or basic charge patches

• Molecular mass and primary amino acid variants including C-terminal lysine clipping

• Secondary through quaternary protein structure and antigen complex variations

• Dimerization and aggregation

• Contamination with host cell proteins

Several modalities of Mass Spectroscopy (MS) analysis have emerged as core technologies for antibody peptide and glycan characterization. The following are some examples:

• ESI, MALDI-TOF, Native, and Hydrogen/Deuterium Exchange MS methods for intact antibody and antibody/antigen complex analysis

• LC-MS and LC-MS/MS utilizing stable isotope labeled peptide internal standards for antibody quantification

• Middle-up and middle-down LC-MS analysis of chemically or enzymatically generated antibody fragments

Dr. Beck and others are investigating not only new methodologies for improving the quality control of existing therapeutic antibodies, but for the development of biosimilars, biobetters, and next generation antibodies.2 In his recent review article, Dr Beck highlights two new enzymes from Genovis Enzyme Technologies for antibody-specific glycan and peptide analysis:3

• FabRICATOR® IdeS Protease

• IgGZERO™ (EndoS Glycanase)

FabRICATOR IdeS Protease for Improved Fc Fragment GenerationPeptide hydrolysis of antibodies has historically utilized proteases such as papain, pepsin, and endoproteinase Lys-C, which can suffer from limited specificity. Recently, the immunoglobulin-degrading IdeS cysteine protease (FaBRICATOR) from Streptococcus pyogenes has provided an improvement in specificity and digestion time. Since FabRICATOR cleaves under the hinge domain, antibodies with glycosylated Fab regions will yield glycans associated with two separate peptide fragments. This enables more effective glycan analysis, particularly relating to the effector functions of Fc fucosylation and galactosylation.4

Because of its exceptionally high specificity for IgG protein sequences, Goetze, A.M. et al. were able to utilize the FaBRICATOR enzyme to cleave IgG in serum to produce Fc fragments for analysis by LC-MS to identify individual haplotypes.5


Advantages of using FabRICATOR for F(ab’)2 or Fab’ production• Faster digestion times – IgG cleaved within 30 minutes compared to

digestion times as long as 12 hours for pepsin and papain.

• Optimized formulation and protocol specific for IgG fragmentation

• High specificity compared to other proteases yielding the same fragment under variable conditions.

• Digestion performed under mild conditions at physiological pH with no negative effect on immunoreactivity of the generated fragments.

• Antibodies Compatible with FabRICATOR enzyme: Human IgG, Humanized IgG, Chimeric IgG, Monkey, Rabbit, Sheep, Mouse IgG2a, and Mouse IgG3

Figure 1.

FabRICATOR cleaves IgG isotypes just below the hinge region, generating an intact F(ab’)2 fragment and a Fc fragment.

New Enzymes for Antibody Characterization from Sigma-Aldrich | Volume 8 No. 2

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FabRICATORCleavage Site




PAPELLG GP

FabRICATOR

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FabRICATOR

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PAPELLG GP

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Figure 2.

Cleavage of a therapeutic antibody with FabRICATOR just below the hinge region, generating an intact F(ab’)2 fragment and a Fc fragment.8

This provides simplified MS analysis of antibodies that have two different glycosylation sites on the heavy chain, by yielding two separate peptide fragments, each with one glycosylation site.

IgGZERO (EndoS Glycanase)Enzymatic glycan hydrolysis of antibodies has historically utilized glycanases such as PNGase F, and other endo and exoglycosidases. IgGZero (EndoS), also from Streptococcus pyogenes, has demonstrated exceptional hydrolytic activity specific for IgG bound glycans.6,7 The enzyme cleaves the chitobiose core of the glycan on IgG from various sources, such as human, rabbit, mouse, Rhesus monkey, goat, sheep, rat, horse, dog, porcine, and more.

Advantages of IgGZERO:• IgGZERO is the first known bacterial enzyme with a unique specificity

for native IgG, no denaturation of the substrate is required. The activities of other endoglycosidases, such as PNGase F, require or are enhanced by denaturation of the glycoprotein substrate.

• Removal of IgGZERO is extremly easy as the recombinant enzyme contains an

• N-terminal histidine tag.

• IgGZERO efficiently cleaves bound IgG from IgG Fc receptors without producing additional damage to native cells.

23500 23750 24000 24250 24500 24750 25000 25250 25500 25750 26000

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275272722127059

2689626668

27690

2807327709 28218

scFc Domain

Fd' Domain

mass

mass

FabRICATOR® - treated Therapeutic Antibody


Figure 3.

IgGZERO shows specific endoglycosidase activity on native IgG because it hydrolyzes the conserved glycans attached to Asp297 on IgG heavy chains.8

Figure 4.

Cleavage sites of IgGZERO and PNGase F

450000

%

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46000 47000 48000 49000 50000 51000 52000

450000

%

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46000 47000 48000 49000 50000 51000 52000

450000

%

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IgG2a Heavy Chain

PNGase F30 min

IgGZERO30 min

50554 50714

49460

50554

49108

50714

SummaryTherapeutic antibody analysis is evolving, particularly as liquid chromatography, electrophoresis, and MS workflows are being refined. FabRICATOR and IgGZero enzymes are adding another dimension of accuracy and ease-of use to these methods.

Find out more about products for antibody characterization including the Genovis enzymes, and Maxi and Minispin Columns on our Antibody Characterization Website.

β1,4 β1,2

α1,6α1,6

α1,3β1,4

β1,4 β1,4

β1,2PNGase FIgG ZERO

Asn

Gal GlcNAcMan Fuc

Key Products

Cat. No. Product Description07298 FabRICATOR® for cleaving 2 mg IgG, 2000 U/vial 8

77661 FabRICATOR 8 for cleaving 5 mg IgG, 5000 U/vial

42607 FragIT™ MaxiSpin 8 for cleaving 10-100 mg IgG, 1 column

94733 FragIT MaxiSpin Kit 8 for cleaving and purifying up to100 mg IgG

00283 FragIT MicroSpin 8 for cleaving up to 0.5 mg IgG, 2 columns

41476 FragIT MicroSpin 8 for cleaving up to 0.5 mg IgG, 5 columns

42606 FragIT MicroSpin 8 for cleaving up to 0.5 mg IgG,10 columns

48776 FragIT MicroSpin Kit 8 for cleaving and purifying up to5 x 0.5 mg IgG

03064 FragIT MicroSpin Kit 8 for cleaving and purifying up to0.5 mg IgG

89379 FragIT MidiSpin 8 for cleaving 1-10 mg IgG, 1 column

42605 FragIT MidiSpin Kit 8 for cleaving and purifying up to10mg IgG

IgGZero Cat. No. Product Description96395 deGlycIT MaxiSpin 8 deglycosylates 10-100 mg IgG,

1 column

68763 deGlycIT MicroSpin 8 deglycosylates up to 0.5 mg IgG,2 columns

90308 deGlycIT MicroSpin 8 deglycosylates up to 0.5 mg IgG,5 columns

04251 deGlycIT MicroSpin 8 deglycosylates up to 0.5 mg IgG, 10 columns

67533 deGlycIT MidiSpin 8 deglycosylates 1-10mg IgG, 1 column

36111 IgGZERO™ 8 for cleaving 1 mg IgG, 1000 U/vial

94509 IgGZERO 8 for cleaving 5 mg IgG, 5000 U/vial



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©2013 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Where bio begins is a trademark of Sigma-Aldrich Co. LLC. Sigma brand products are sold through Sigma-Aldrich, Inc. SAGE and Prestige Antibodies are registered trademarks of Sigma-Aldrich Co. LLC. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip. FabRICATOR is a registered trademark of Genovis AB. IgGZERO is a trademark of of Genovis AB.

References

1. Goetze, A.M. et al., Assessing monoclonal antibody product quality attribute criticality through clinical studies, MAbs, 2, 500–507 (2010).

2. Beck, A. et al., Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry, Anal. Chem., 84(11), 4637–46 (2012).

3. Beck, A. et al., Anal. Chem., Special Issue: Fundamental and Applied Reviews in Analytical Chemistry, 2013.

4. Ryan, M.H. et al., Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid, Mol. Immunol., 45, 1837–1846 (2008).

5. Goetze, A.M. et al., Rapid LC-MS screening for IgG Fc modifications and allelic variants in blood, Mol. Imm., 49, 338–352 (2011)..

6. Goodfellow, J.J. et al., An endoglycosidase with alternative glycan specificity allows broadened glycoprotein remodelling. J. Am. Chem. Soc., 134(19), 8030–8033 ( 2012).

7. Goetze, A.M. et al., Rates and impact of human antibody glycation in vivo. Glycobiology, 22(2), 221–234 (2011).

8. Sigma-Aldrich data


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Your Biology Resource - Sigma-Aldrich · PDF fileYour Biology Resource Volume 8 ... PAPELLG GP FabRICATOR 119 120 PAPPVA GP FabRICATOR ... MAbs, 2, 500–507 (2010). 2. Beck, A. et