Site‐specific monitoring of N‐Glycosylation profiles of a CTLA4‐Fc‐fusion protein from the secretory pathway to the extracellular environment

Yang

Biotech & Bioengineering

et al. 2019

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N-and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N-and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc Nglycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi-and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N-and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest. K E Y W O R D S CHO-GS cells, culture media, EPO-Fc protein, intact glycopeptide analysis, site-specific glycosylation SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section.

Section: Discussionmentioning

confidence: 99%

Characterization of intact glycopeptides reveals the impact of culture media on site‐specific glycosylation of EPO‐Fc fusion protein generated by CHO‐GS cells

Yang

Biotech & Bioengineering

et al. 2019

J. Am. Soc. Mass Spectrom.

“…In-source fragmentation was minimized by optimizing source parameters through a design of experiments approach, extending work by Barton and co-workers who used a one factor at a time approach. 48 Here, we generate a well-defined model of the parameter space, allowing the interdependencies of source parameters to be assessed and conditions to be determined that minimize in-source fragmentation while maintaining sensitivity. Differential ionization was addressed by determining normalization factors to correct for bias in signals owing solely to the nature of glycoform.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Transitions corresponding to these and other observed glycoforms were added to the MAM method, and studies were performed to bridge this approach to the conventional method. In-source fragmentation was minimized by optimizing source parameters through a design of experiments approach, extending work by Barton and co-workers who used a one factor at a time approach . Here, we generate a well-defined model of the parameter space, allowing the interdependencies of source parameters to be assessed and conditions to be determined that minimize in-source fragmentation while maintaining sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation

Hines

Edgeworth

Devine

et al. 2023

Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography–mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.

“…Thus, the number of antennas and number of sialic acid residues in a glycoprotein play an important role in the clearance of CTLA4-Ig fusion protein (Jing et al, 2010). CTLA4-Ig primarily has two-to tri-antennary structures in the CTLA4 region (Bora de Oliveira et al, 2017). Therefore, a recent study investigated the cooverexpression of GnT and α2,6-ST as a means to produce a highly sialylated CTLA4-Ig fusion protein (Lim et al, 2017;Lim et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…We termed the recombinant human CTLA4-Ig protein, which has four antennas terminally capped with sialic acid, "ST6." It should be noted that the N-glycan structure of the CTLA4 region of ST6 is different from that of abatacept, which mainly consists of bi-antennary (GnT-II) and tri-antennary (GnT-III) structures (Bora de Oliveira et al, 2017). The overexpression of α2,6-ST serves to enhance the sialylation of various glycoproteins, while the overexpression of α2,6-ST serves to mediate anti-inflammatory effects and facilitate human-like glycosylation (Chung et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

A Novel Therapeutic Effect of a New Variant of CTLA4-Ig with Four Antennas That Are Terminally Capped with Sialic Acid in the CTLA4 Region

Piao

Yun

Kim

et al. 2022

Biomol Ther (Seoul)

Rheumatoid arthritis (RA) is a multifactorial immune-mediated disease, the pathogenesis of which involves different cell types. T-cell activation plays an important role in RA. Therefore, inhibiting T-cell activation is one of the current therapeutic strategies. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), also known as abatacept, reduces cytokine secretion by inhibiting T-cell activation. To achieve a homeostatic therapeutic effect, CTLA4-Ig has to be administered repeatedly over several weeks, which limits its applicability in RA treatment. To overcome this limitation, we increased the number of sialic acid-capped antennas by genetically engineering the CTLA4 region to increase the therapeutic effect of CTLA4-Ig. N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST) were co-overexpressed in Chinese hamster ovary (CHO) cells to generate a highly sialylated CTLA4-Ig fusion protein, named ST6. The therapeutic and immunogenic effects of ST6 and CTLA4-Ig were compared. ST6 dose-dependently decreased paw edema in a mouse model of collagen-induced arthritis and reduced cytokine levels in a co-culture cell assay in a similar manner to CTLA4-Ig. ST6-and CTLA4-Ig-induced T cell-derived cytokines were examined in CD4 T cells isolated from peripheral blood mononuclear cells after cell killing through irradiation followed by flow-and magnetic-beadassisted separation. Interestingly, compared to CTLA4-Ig, ST6 was substantially less immunogenic and more stable and durable. Our data suggest that ST6 can serve as a novel, less immunogenic therapeutic strategy for patients with RA.