Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies

Abstract

The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.

Introduction

Monoclonal antibodies (mAbs) have been widely used for the treatment of many diseases, including cancers, autoimmune disorders, and inflammatory and infectious diseases [1], [2]. Besides target recognition by the Fab moiety, the Fc region of IgG antibodies mediates immune responses through its interaction with Fc receptors on the immune cells [3], [4], [5]. The glycans on the Fc region of IgG, especially at position Asn-297, are typically heterogeneous biantennary complex-type N-glycans with significant structural diversity [6]. Moreover, the Fc glycan of IgG affects its interaction with Fc receptor and, thereby, influences the immune-response activities (effector functions) [7], [8], [9], [10]. Therefore, various glycoengineering technologies have been developed to prepare better-defined glycoforms with no core fucosylation to enhance the interaction with Fc receptor (FcγRIIIa) and improve antibody dependent cellular cytotoxicity (ADCC) [11], [12], [13].

Obinutuzumab (GA101) and mogamulizumab (POTELLIGENT), FDA-approved glycoengineered antibodies, are mixtures of antibody glycoforms without core fucosylation. [14], [15], [16]. Current antibody productions either from natural or recombinant protein expression system typically generate heterogeneous mixtures of glycoforms. To pursuit homogeneous antibody glycoform, endoglycosidase-catalyzed antibody glycoengineering has become a powerful synthesis or identification tool [17], [18], [19], [20]. Generally, the procedure involves cleaving most of the N-glycans by the wild-type (WT) endoglycosidases but keeping the innermost GlcNAc residue on the glycosylation site to form the monoglycosylated antibody IgG-GlcNAc. Then, an endoglycosidase mutant with reduced glycosidase activity is used to transfer a synthetic N-glycan building block with oxazoline to the IgG-GlcNAc. This chemoenzymatic method requires isolation of IgG-GlcNAc from endoglycosidase and fucosidase after the deglycosylation step and separation of the final glycoengineered product from the endoglycosidase mutant after the transglycosylation step. The large quantity of fucosidase used in IgG defucosylation could pose separation problems, and the residual contamination of the endoglycosidase could hydrolyze the final product.

Enzyme was immobilized onto a solid support to preserve catalytic performance for a target reaction [21]. The use of agarose bead immobilization has been described in the industrial antibody manufacturing. The use of magnetic beads for immobilization has been described in the development of diagnostics and therapeutics, including DNA-extraction [22], cell manipulation and cellular-specific targeting, magnetic resonance imaging (MRI) [23], [24], [25], and targeted medication [24]. In many cases, using magnetic beads instead of agarose beads improves binding efficiency. For example, the non-porous magnetic beads were amino-functionalized to enhance the binding in immobilizing nucleic acids or proteins (enzymes, streptavidin, and antibodies, etc.) [26].

We developed the first glycoengineering method that immobilized both EndoS and fucosidase enzymes on magnetic beads to enhance the activity of antibodies. We chose magnetic beads based because of their good biocompatibility, cost effectiveness, and easy separation by magnet without filtration; moreover, with size < 1 µm, their large surface-to-volume ratios convey higher catalytic efficiency [27].

In this paper, we reported two glyco-engineering and antibodies separation approaches. The first is to covalently immobilize wild type endoglycosidase S (EndoS) [28], fucosidase [6], and EndoS2 mutant [29] on magnetic beads through disuccinimidyl suberate (DSS) or succinimidyl iodoacetate (SIA) linker. We found that enzymes immobilized on magnetic bead B through the DSS linker was the most efficient for antibody IgG glycoengineering.

The second approach is to covalently immobilized fucosidase and biotinylated wild type EndoS2 and EndoS2 mutant on magnetic beads to obtain homogeneous antibody glycoforms sequentially by removing endoglycosidase and fucosidase without the need to purify the deglycosylated antibody intermediate. Because the enzymes immobilized on magnetic beads can be easily separated by magnet after glyco-engineering, the cost is significantly reduced as the fucosidase can be recovered and reused. In addition, the highly specific and rapid on-rate association and strong affinity of streptavidin to biotin (with dissociation constant K_d in the order of 4 × 10^-14 M) would complete the isolation of biotinylated enzymes without any residual enzymes [30]. After the immobilized fucosidase on magnetic beads were recovered, the streptavidin coated magnetic beads were used to remove the biotinylated EndoS2 and its mutants after the deglycosylation and transglycosylation steps without the column purification steps. We also demonstrated that both approaches are effective in large-scale production and isolation of antibodies.