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. 2015;7(4):719-31.
doi: 10.1080/19420862.2015.1046663.

A chemical and computational approach to comprehensive glycation characterization on antibodies

Ramsey A Saleem  1 , Brittany R Affholter, Sihong Deng, Phil C Campbell, Kelli Matthies, Catherine M Eakin, Alison Wallace
Affiliations

A chemical and computational approach to comprehensive glycation characterization on antibodies

Ramsey A Saleem et al. MAbs. 2015.

Abstract

Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS(2) mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS(1) based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur.

Keywords: BA, boronate affinity chromatography; CDR3, complementary-determining region 3; CEX, cation exchange chromatography; CID, collision induced dissociation; CV, coefficient of variation; Da, daltons; EIC, extracted ion chromatogram; HC-CDR3, heavy chain complementary determining region 3; HPLC, high performance liquid chromatography; LC-MS2, liquid chromatography coupled with tandem mass spectrometry; MS1, a mass to charge ratio survey scan; MS2, tandem mass spectrometry - selected ions from MS1 are fragmented and fragment ion mass measured; UPLC, ultrahigh performance liquid chromatography; boronate affinity chromatography; glycation; mAb, monoclonal antibody; structural modeling; targeted mass spectrometry; Å, angstroms.

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Figures

Figure 1.
Figure 1.
This mAb is glycated and the glycation enriches in the acidic CEX fraction. (A) Intact mass analysis of the deglycosylated mAb shows that glycation increases from early development (15% glycation) to late stage (18%) production processes. The glycated species is indicated by light blue shading while the non-glycated population is indicated by light purple shading. (B) Boronate affinity estimates of glycation levels are in agreement with the intact mass analysis at 19%. (C) Intact mass analysis of the CEX fractions shows that glycation is enriched in the acidic fraction. Estimates of purity, potency, peak height and glycation level are shown for each fraction in the table.
Figure 2.
Figure 2.
Characterization of the mAb Glycation by MS2 peptide mapping and FabRICATOR-Intact Mass analysis. (A) A cartoon of cleavage fragments generated by treatment with FabRICATOR and reduction (left panel). Intact mass analysis of the FabRICATOR fragments (right panel) shows glycation (light blue shading) enriching in the Fd portion of the Acidic CEX fraction. (B) Searches of the MS2 data using the Sequest search engine identify one peptide in the light chain with an XCorr score of 2.93. The glycated lysine residue is shown by the bold K. Inset shows a typical neutral loss peak that arises in the MS2 spectra precluding identification of the ion series. Labeled spectra were overlaid with equivalent intensity, thicker lines, to enhance visibility.
Figure 3.
Figure 3.
Sodium borohydride derivatization facilitates the identification of MS2 ion series. (A) Mechanism of action of NaBH derivatization, where the double bond between the reducing sugar and the nitrogen of the lysine residue is reduced. (B) MS2 spectra of the derivatized peptide showing the differences between derivatized and underivatized (inset) samples. Labeled spectra were overlaid with equivalent intensity, thicker lines, to enhance visibility. (C) A cartoon of sites of glycation identified by derivatization of glycation and spectral searching for peptides with derivatized glycan mass shifts. Approximate sites of glycation are shown by red arrows and the numbers correspond to numbers identified in Table 2.
Figure 4
Figure 4
(See previous page). Forced glycation of the mAb reveals a decrease in potency and an increase in glycation of K99. (A) Intact mass of the forced glycated mAb. On the top is the mAb incubated with 5% glucose while below is the mAb incubated with 5% sorbitol, a negative control. (B) Potency, expressed here as the relative potency of the mAb in glucose over the mAb in sorbitol. By day 6, potency has decreased to ˜82%. (C) Base Peak and survey MS1 scans of the Base Peak area for the glycated form of the peptide. Note that in the late stage process molecule, the +2 and +3 forms of the ions co-elute with another ion species (the 726 m/z species). The sum of the relative intensity of the 613 m/z (+3) and 919 m/z (+2) ions is estimated to be 36% of the 726 m/z ion. This estimate is used to adjust the estimate of the area for the glycated form.
Figure 5.
Figure 5.
Structural modeling of this mAb reveals a putative mechanism for mediation of non-enzymatic glycation. (A) A three dimensional ribbon model of the mAb. The heavy chain is shown in green while the light chain is shown in gray. Lysine residues are shown in blue, while glycated lysine residues are shown in burgundy. (B) Two views of the K99 glycation hot spot. Two residues on the heavy chain, D98 and D105, and one residue on the light chain, E61, are able to exert an effect on K99. The red coloring on D98, D105 and E61 shows the position of oxygen atoms in the carboxylic acid.
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