Antibody Fc-glycan Analysis by FabRICATOR® Digestion and LC-MS

Using the FabRICATOR (IdeS) enzyme for antibody subunit analysis has become a standard analytical method for rapid characterization of monoclonal antibodies (mAbs). FabRICATOR digests IgG below the hinge, generating F(ab’)2 and Fc/2 fragments. The unique specificity of the enzyme has led to the development of a range of analytical strategies for middle-level characterization of therapeutic mAbs and related products, such as antibody-drug conjugates (ADCs), bispecific antibodies and Fc-fusion proteins. The single FabRICATOR digestion site and the following accuracy of the mass profile have enabled detection of the antibody glycosylation profile and other critical quality attributes. Here, the Fc glycosylation profile of the therapeutic antibody trastuzumab was analyzed using FabRICATOR in combination with LC-MS.

The antibody was digested with FabRICATOR, denatured and reduced, and the resulting Fc/2, Fd’ and light chain (LC) fragments were separated and analyzed by LC-MS. The short digestion time of FabRICATOR in combination with the ~25 kDa Fc/2 fragment, enabled a fast analysis of the glycan profile with monoisotopic resolution when analyzed by LC-MS. The presented workflow is robust and can be used to study not only glycosylation, but also critical quality attributes such as oxidation, glycation and sequence identity.

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Summary

  • The Fc glycosylation profile of trastuzumab was determined with high accuracy compared to theoretical values
  • FabRICATOR (IdeS) enables a rapid and easy platform method for IgG fingerprinting
  • FabRICATOR specifically digests IgG below the hinge within 30 minutes
  • Detailed methods for separation and mass spectrometry analysis at the middle-level are described

Fabricator LC-MS

Introduction

Recombinant mAbs for therapeutic use have been developed as effective drugs against various diseases, such as cancer and autoimmune diseases. A critical quality attribute of therapeutic antibodies is the glycosylation profile, since it may affect prominent features like stability and solubility as well as pharmacodynamic and pharmacokinetic properties. The glycosylation process is a part of the posttranslational modification of proteins and close monitoring of this attribute during the bioprocess development and the manufacturing is very important (1,2).

A majority of all commercial therapeutic antibodies are of class IgG and contain an N-glycan in the Fc domain. To measure the Fc glycosylation, a widely accepted method is to apply hydrophilic interaction liquid chromatography-ultra high-performance liquid chromatography (HILIC-UHPLC) on released N-glycans after being labeled with 2-aminobenzamide (2-AB; 1,2). This methodology suffers from several hands-on steps and is time consuming.

An alternative method for glycan profile analysis has been suggested, involving direct analysis of the antibody Fc/2 fragment using LC-MS (2,3). The cysteine protease FabRICATOR (IdeS) digests human IgG at a specific site below the hinge, generating a homogenous pool of Fc/2 and F(ab’)2 fragments (4; Fig. 1). The smaller fragment size of Fc/2 (~25 kDa) compared to the intact heavy chain (HC, ~50 kDa), allows for monoisotopic resolution of the glycan profile when analyzed with ESI-MS. Digestion using FabRICATOR is rapid, 30 minutes, and due to its specific activity, there is no risk of overdigestion that might otherwise result in heterogeneity of the antibody sample (2). In an extensive comparison between methods for Fc glycosylation analysis, the middle-up analysis of Fc/2 glycans was found to be robust and to provide reliable results for the IgG glycoprofile (5).

This application presents a workflow for evaluating the glycan profile of the therapeutic human IgG1 trastuzumab using FabRICATOR in combination with LC-MS analysis. The measured monoisotopic masses of Fc/2, LC and Fd’ fragments are compared to theoretical data and the relative distribution of the different Fc glycoforms is calculated.
LC-MS Fabricator

Figure 1. The IgG is digested with FabRICATOR followed by a denaturation and reduction step for LC-MS analysis.

Materials & Methods

Antibody Digestion using FabRICATOR

2000 units of FabRICATOR was reconstituted in 30 μl Milli-Q water (MQ) to a final concentration of 67 U/μl. To obtain a final ratio of 1 unit enzyme per 1 μg of antibody, 4 μl trastuzumab (25 mg/ml) and 1.5 μl FabRICATOR (67 U/μl), was added to 44.5 μl PBS to a final IgG concentration of 2 mg/ml. The sample was then incubated at 37°C for 30 minutes (for an overview of the workflow, see Fig. 2).

 

Fabricator visual LC-MS

Figure 2. FabRICATOR was reconstituted in MQ before added to IgG in PBS for digestion.

Denaturation and Reduction of Antibody Subunits

6 μl of the FabRICATOR-digested IgG sample (2 mg/ml), was added to 10 μl 8 M guanidine hydrochloride (GdHCl) and 4 μl 0.5 M DTT (fresh) to final concentrations of 0.6 mg/ml IgG, 4 M GdHCl and 100 mM DTT. This prepared sample was then denatured and reduced by incubation at 37°C for 60 minutes prior to LC-MS analysis. An overview of the FabRICATOR sample preparation for LC-MS analysis is given in Fig. 1.

Analysis by Liquid Chromatography and Mass Spectrometry

The prepared IgG sample was analyzed using LC-MS. To avoid sample buffer exchange prior to MS analysis, a 13 minutes desalting step was added before an acetonitrile/water gradient was applied to a BEH C4 column from Waters according to Table 1 and Table 2. During the desalting step, the valve was switched to waste. The separated IgG fragments were then analyzed by ESI-Q-TOF MS on a Bruker Impact II instrument under conditions given in Table 3. The obtained TIC data were deconvoluted using the MaxEnt algorithm to determine masses. The theoretical masses were calculated from amino acid sequences obtained from the KEGG database (http://www.genome.jp/kegg/).

 

Table 1. Liquid Chromatography Setup

LC system

Agilent 1290 Infinity

Sample temp.

8°C

Flow rate

0.2 ml/min

Injection volume

1.7 μl

Sample load

1 μg

Column

Acquity UPLC Protein column, BEH C4, 300Å, 1.7 μm,
2.1 mm x 100 mm column (Waters)

Column temp.

50°C

Method time

42 minutes

 

Table 2. Liquid Chromatography Method

RP-LC solvent gradient

Time (min)

Solvent A (%): 0.1% formic acid in MQ

Solvent B (%): 0.1% formic acid in 95% ACN: 5% MQ

0

90

10

13

90

10

14

75

25

26

54

36

29

10

90

32

10

90

32.01

90

10

42

90

10

 

Table 3. Mass Spectrometry Conditions

Instrument

Bruker Impact II

Analysis method

ESI-Q-TOF MS

Resolving power

50 000

Mass range

300 - 3000 m/z

ESI source voltage

4.5 kV

ESI source temp.

220°C

Nebulization gas pressure

1.8 Bar

Nebulization gas ow rate

8.0 l/min

Data analysis program

Compass DataAnalysis 2.0 (Bruker)

Deconvolution algorithm

MaxEnt

Results & Discussion

After denaturation and reduction, the FabRICATOR-digested trastuzumab fragments eluted as three protein peaks detected with UV 280 when separated by RP-LC (Fig. 3).

 

Figure 3. HPLC chromatogram (UV 280 signal) of FabRICATOR-digested trastuzumab after denaturation and reduction. The three fragments Fc/2, LC and Fd’ are depicted in the chromatogram.

 

Mass determination of the three peaks with ESI-Q-TOF MS followed by analysis of the deconvoluted TIC data with the MaxEnt algorithm, confirmed that the mass of the peaks corresponded to theoretical values of the reduced forms of the Fc/2, Fd’ and LC fragments, with a high mass accuracy (Δppm < 2; Fig. 4-6 and Table 4). The monoisotopic masses of the four main trastuzumab Fc/2 glycoforms were determined and their relative intensities were calculated (Fig. 4 and Table 5).
Figure 4. Deconvoluted mass spectrum of trastuzumab Fc/2 fragments after FabRICATOR digestion, denaturation and reduction. The different glycoforms of Fc/2 are depicted in the spectrum.

 

deconvoluted LC-MS

 

Figure 5. Deconvoluted mass spectrum of trastuzumab Fd’ fragment after FabRICATOR digestion, denaturation and reduction.

 

LC-MS deconvoluted

 

Figure 6. Deconvoluted mass spectrum of trastuzumab LC fragment after FabRICATOR digestion, denaturation and reduction.

 

Table 4. Mass Determination of Detected Peaks

Fragment

Detected Mass (Da)

Theoretical Mass (Da)

Mass Δ(ppm)

Fc/2 (240-449) + G0F

25220.5079

25220.4634

1.8

Fc/2 (240-449) + G1F

25382.5574

25382.5162

1.6

Fc/2 (240-449) + G2F

25544.6034

25544.5690

1.3

Fc/2 (240-449) + G0

25074.4503

25074.4055

1.8

Fd’ (1-239)

25367.4781

25367.5174

-1.5

LC

23428.5130

23428.5238

-0.5

 

Table 5. Relative Intensities of Fc/2 Glycoforms of Trastuzumab

Fc/2 Glycoforms

Relative intensity (%)

G0

4.8

G0F

44.7

G1F

42.7

G2F

7.9

 

The relative glycoform distribution was found to correlate well with data from other methods for glycan analysis, such as HILIC-UHPLC separation of released and 2-AB labeled glycans as well as LC- MS analysis of reduced, intact antibody (6,7). The sample preparation prior to LC-MS analysis included digestion of the antibody using FabRICATOR for 30 minutes, followed by denaturation and reduction of the antibody fragments for 60 minutes. Buffer exchange prior to MS analysis was avoided by incorporating a desalting step in the HPLC gradient. The denaturation and reduction steps can be excluded if there is no interest in the Fd’ and LC fragments.

 

In conclusion, we here present a fast and easy workflow to determine the glycan composition of therapeutic antibodies, and the same workflow can be utilized to analyze several critical quality antibody attributes (2). This method is useful within both development and production of mAbs, for example to control the glycan profile during process development and to assure batch-to-batch consistency.

References

  1. Reusch D., Haberger M., Maier B., Maier M., Kloseck R., Zimmermann B., Hook M., Szabo Z., Tep S., Wegstein J., Alt N., Bulau P., and Wuhrer M. (2015). Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles – Part 1: Separation- based methods, mAbs, 7:1, 167-179, doi: 10.4161/19420862.2014.986000.
  2. Sjögren J., Olsson F., Beck A. (2016). Rapid and improved characterization of therapeutic antibodies and antibody related products using IdeS digestion and subunit analysis.Analyst, 141, 3114, doi: 10.1039/c6an00071a.
  3. Chevreux G., Tilly N., and Bihoreau N. (2011). Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry.
    Anal. Biochem. 415, 212–214.
  4. von Pawel-Rammingen U., Johansson B., Björck L. (2002). IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. The EMBO Journal, vol. 21 No. 7 pp. 1607-1615.
  5. Reusch D., Haberger M., Falck D., Peter B., Maier M., Gassner J., Hook M., Wagner K., Bonnington L., Bulau P., and Wuhrer M. (2015). Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles – Part 2: Mass spectrometric methods, mAbs, 7:4, 732-742. doi: 10.1080/19420862.2015.1045173.
  6. Xie H., Chakraborty A., Ahn J., Qing Yu Y., Dakshinamoorthy D. P., Gilar M., Chen W., Skilton St. J., Mazzeo J. R. (2010). Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies. Jandes Bioscience mAbs 2:4, 379-394.
  7. Damen C. W.N., Chen W., Chakraborty A. B., van Oosterhoutm M., Mazzeo J. R., Gebler J. C., Schellens J. H. M., Rosling H., Bejnen J. H. (2009). Electrospray Ionization Quadrupole Ion-Mobility Time-of-Flight Mass Spectrometry as a Tool to Distinguish the Lot-to-Lot Heterogeneity in N-Glycosylation Profile of the Therapeutic Monoclonal Antibody Trastuzumab. J Am. Society of Mass Spectrometry 20(11), 2021-33. doi:10.1016/j.jasms.2009.07.017.

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