Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation

Abstract

Glycosylation can be a critical quality attribute in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity, and pharmacokinetics of therapeutic monoclonal antibodies (mAbs), and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream-downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc γ-based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space to establish holistic and consistent manufacturing processes for the production of antibody therapies.

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

“…In most cases, the developers of innovative therapeutics will keep their manufacturing process information as proprietary. Therefore, biosimilars developers will have to develop their own process with likely variations from that of the original developers, while maintaining similarity with the reference products with minimum variations in the critical quality attributes [ 10 , 11 ].…”

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli

“…Cells of different origins have been used to produce N-glycosylated glycoproteins [ 4 ]. Numerous strategies including cell line engineering, as well as the addition of inhibitors for metabolic glycosylation and glycosidase have been developed to reduce glycoprotein N-glycan heterogeneity [ 15 , 16 ]. Synthetic methods including total synthesis, semi-synthesis, and chemoenzymatic synthesis have been developed to obtain homogeneous glycoproteins [ 17 , 18 , 19 , 20 ].…”

“…Synthetic methods including total synthesis, semi-synthesis, and chemoenzymatic synthesis have been developed to obtain homogeneous glycoproteins [ 17 , 18 , 19 , 20 ]. It is, however, still challenging to achieve glycoprotein N-glycan homogeneity [ 15 ], especially in large-scale productions. For example, monoclonal IgG1-type antibodies are an important type of therapeutic glycoproteins that have seen significant increased clinical applications.…”

Study on antibody Fc-glycosylation for optimal effector functions

“…13,20,21 Despite tremendous progress in the genetic engineering of glycosylation in mammalian and nonmammalian cells to produce glycoengineered antibodies, these methods are still limited by complicated protocols, low production yield, and difficult access to homogenous glycoforms. 22,23 An alternative powerful method to edit the glycosylation pattern is to use an endoglycosidase mediated in-vitro glycoremodeling approach, where the intact heterogeneously glycosylated antibody is first deglycosylated to generate GlcNAc at Asn297, then a new glycan is added enzymatically in a stepwise manner or enbloc in the form of oxazoline through transglycosylation to form a homogeneous glycan with a well-defined structure. 24 In this study, as part of our ongoing efforts to optimize the IgG Fc-glycan for desired effector functions, we sought to investigate how the N-glycans, such as high mannose, hybridand complex-type glycans with various degrees of antennae, sialic acid linkage, terminal fucose linkage, bi-secting GlcNAc, modifications of terminal sialic acid etc., modulate the binding of an antibody to FcgRIIIa for ADCC and CDC activity and to FcgRIIa for the vaccinal effect.…”

“…13,20,21 Despite tremendous progress in the genetic engineering of glycosylation in mammalian and non-mammalian cells to produce glycoengineered antibodies, these methods are still limited by complicated protocols, low production yield, and difficult access to homogenous glycoforms. 22,23 An alternative powerful method to edit the glycosylation pattern is to use an endoglycosidase mediated in-vitro glycoremodeling approach, where the intact heterogeneously glycosylated antibody is first deglycosylated to generate GlcNAc at Asn297, then a new glycan is added enzymatically in a stepwise manner or en-bloc in the form of oxazoline through transglycosylation to form a homogeneous glycan with a well-defined structure. 24…”