March 15, 2015 (Vol. 35, No. 6)
Steven B. Richardson senior scientist, R&D cell- sciences-and-development SAFC
Relying on Simple Supplementation for Improved Glycan Profiles
Over the past decade, notable advances in the control of post-translational modifications to enhance therapeutic properties have continued to drive the development of many next-generation biopharmaceuticals. Glycosylation, the covalent attachment of carbohydrates to organic molecules, is an essential post-translational modi?cation that can affect a wide range of characteristics of therapeutic proteins.
Carbohydrates in the form of asparagine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins. The type, number, and location of oligosaccharides on a therapeutic protein can impact its stability, efficacy and immunogenicity within the body (Figure 1).
Due to the pharmacokinetic effects of the various glycan structures, most therapeutic proteins must have a defined distribution of oligosaccharides to function as intended in vivo. Maintaining a consistent and comparable glycosylation distribution is an integrated development process involving cell-line engineering, clone selection, media development, and bioreactor optimization.
The subsequent scaleup and transfer of a biomanufacturing process can introduce a range of glycosylation patterns for any given protein, thus making it challenging to achieve the targeted product quality profile. This challenge has translated into an unmet need for in-line solutions that enable directional control of critical quality attributes.
Recognizing that improvement in the control of the glycosylation pathway would have a significant impact on the manufacture of biosimilars, as well as branded biopharmaceuticals (particularly next-generation biologics), SAFC embarked on a program to identify critical raw materials for the targeted optimization of the glycosylation process.
Cell culture media optimization plays a significant role in achieving optimal yield and product quality in any culture system. Researchers at SAFC performed a proprietary screen of chemically defined feeds to identify raw materials correlated to culture performance, and specifically those that rescue N-linked glycosylation profiles of therapeutic proteins from selected high-producing clones (Figure 2).
The goal of the research was to establish cGMP-ready, chemically defined supplements that significantly and reproducibly adjust glycan moieties. EX-CELL Glycosylation Adjust (Gal+) was the result of this research. It is a protein-quality supplement (PQS) designed for biological drug production, including biosimilars. PQS enables the desired N-linked glycosylation by increasing the galactose site occupancy on the oligosaccharide to a higher level, and achieves functionally relevant shifts in N-linked glycosylation efficiently. The supplement is titrated into the bioreactor, eliminating the need for trial and error optimization.
Recommendations for Use
As a general rule, culture supplementation with EX-CELL Glycosylation Adjust (Gal+) should be initiated at 0.2% (v/v) beginning on day 2 and then continued every other day up to day 10 of a two-week fed-batch culture. It is strongly suggested that a titration be performed to determine the optimal concentration for a specific process. The titration should start in the 0.5X to 2X range and expand if necessary.
For the initial raw-material screening runs and testing of the new supplement, a TPP bioreactor tube production process was employed. In each case, a 14-day fed-batch culture was initiated at a seeding density of 0.3 -0.5 x 106 vc/mL in 25 mL of proprietary chemically defined media. The TPP tubes were incubated at 37 ± 1°C in an atmosphere of 5% CO2 and 80–85% relative humidity with agitation at 200 rpm.
The cell culture was fed at 5–10% (v/v) beginning on day 3 and repeated every other day up to day 11 of the two-week fed-batch culture. Samples were removed as appropriate for various analyses, including cell counting, to monitor growth and viability, and bioprofiling to monitor metabolites. The supernatant and spent media were also evaluated to determine the productivity level and state of the culture system. The relative glycan distribution was analyzed via size-exclusion chromatography-mass spectrometry.
SAFC has evaluated the performance of the new supplement in a broad range of cell lines, including its CHOZN® cell-line platform and CHO-GS, CHO-M, DuxB11, and NS0 lines. Supplemented fed-batch production processes have demonstrated a twofold to fourfold increase in relative G1F and G2F distributions when compared with processes without supplementation. The results for three different mAb-producing CHO cell lines are presented in Table 1.
It should be noted that while the glycosylation profile was dramatically increased in all three cases, the use of the EX-CELL Glycosylation Adjust (Gal+) protein-quality supplement did not have a negative effect on the desirable process outputs. In all three examples, identical or higher cell densities and volumetric productivities were obtained, as can be seen in Table 1.
With this high level of cross-functionality, it is possible to rescue processes that suffer from poor glycosylation profiles and develop the data required for regulatory approvals, particularly for biosimilars.
In summary, to address the significant unmet need in the biopharmaceutical industry for a practical method for improving glycosylation profiles, SAFC performed an extensive multivariate analysis of critical raw materials to identify those that have an optimum influence on glycan attributes. The newly developed protein-quality supplement EX-CELL Glycosylation Adjust (Gal+) provides a twofold to fourfold increase in the relative G1F and G2F distributions for a wide range of bioproduction cell origins, including CHO-GS, CHO-M, DuxB11, and NS0 lines, with minimal effect on the volumetric productivity and viable cell density.