Optimal Synthetic Glycosylation of a Therapeutic Antibody

Abstract

Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.

Keywords: antibodies; endoglycosidases; glycoengineering; glycosylation; native mass spectrometry.

Figure 1

a) Endoglycosidase‐catalyzed glycosylation with activated sugar donors may lead to competing chemical glycation. b) Current mAbs are formed as mixtures of glycoforms; G0F, G1F, and G2F predominate. c) EndoS‐WT cleaves the core of mixed N‐glycans and can subsequently catalyze glycosylation with oxazoline donors,5a to give, in principle, purer glycoform distributions. d) Payload molecules may also, in principle, be introduced directly through glycosylation or indirectly by the incorporation of reactive handles in sugars and a subsequent selective reaction.

Figure 2

a) EndoS‐WT‐catalyzed deglycosylation of Herceptin gives trimmed Herceptin 1. Subsequent glycosylation with the sugar oxazoline 2 gives remodeled Ab 3. b) Deconvoluted rMS data for samples of reduced commercial, trimmed, and remodeled Herceptin (see Figures S8–S10). c) Native MS for intact commercial, trimmed, and remodeled Herceptin. d) Single‐charge‐state peaks. Colored circles indicate major species present. A minor, non‐glycan +176 Da component (EndoS‐ and PNGaseF‐resistant) was present in all Herceptin (including commercial) samples analyzed.

Figure 3

Treatment of 1 with donor 2 at pH 7.4 in the absence of an enzyme leads to substantial glycation. a) nMS of an intact glycated antibody. The inset is an expansion of the +25 charge state. b) rMS showing that the majority of glycation occurs on the heavy chain.

Figure 4

Native MS of glycosylation products after optimization of the reaction conditions with the native decasaccharide donor 2. The inset shows an expansion of the +25 charge‐state peaks.

Figure 5

a) Circular dichroism analysis shows no gross structural changes during remodeling with unnatural sugar 6 b to form 7 b. b) Native MS of the glycosylation product 7 b after optimization of the reaction conditions with the azide‐tagged decasaccharide donor 6 b. The inset shows an expansion of +25 charge‐state peaks.

Figure 6

Cell‐surface staining of HER2 with 7 b modified with rhodamine cargo. Flow cytometry histograms of a) SK‐BR‐3 cells (HER2(+)) and b) MCF‐7 cells (HER2(−)), unstained (gray) or stained with the synthetic Ab (pink). c) A cumulative distribution function plot of the summed fluorescence intensity shows a 2.3‐fold change in geometric mean intensity for SK‐BR‐3.