Glycosylation Modulation in Fed-batch Production of Therapeutic Proteins
Understanding the Impact of Glycosylation in Monoclonal Antibody Development
Protein glycosylation, the attachment of glycans to proteins, is a key post-translational modification that plays a vital role in determining the efficacy, stability, and immunogenicity of monoclonal antibodies (mAbs) and other recombinant proteins. The addition of sugars like galactose, sialic acid, fucose, and mannose influences the glycosylation profile of mAbs, affecting their therapeutic performance and patient safety (Figure 1).
Figure 1.Glycan structure of monoclonal antibodies: Example of A) terminal galactosylated (G2F), B) terminal sialylated (A2F), and C) terminal N-acetylglucosamine (GlcNAc; G0F).
(Pink diamonds represent sialic acid, yellow circles galactose, blue squares N-acetylglucosamine, green circles mannose, and red triangles fucose.)
When optimizing a production process, both product yield and product quality are crucial factors to consider. The glycosylation profile of a protein is a critical quality attribute that requires stringent control to maintain product consistency. Many factors contribute to the glycosylation profile of a mAb including the cell line, process parameters, media, and feed components. Achieving the targeted glycosylation profile relies on the right combination of medium and feed, as well as a thoughtful feeding strategy, careful consideration of the feed composition, and precise control of culture conditions.
This technical article describes feeding strategies for fed-batch processes using the Cellvento® ModiFeed platform of chemically defined, highly concentrated feeds. This platform offers a comprehensive solution, including the complete feed Cellvento® ModiFeed Prime and three feeds specifically designed to modulate glycosylation profiles of the target protein while not sacrificing performance (Cellvento® ModiFeed Gal+ , Cellvento® ModiFeed Gal-, Cellvento® ModiFeed Sial+).
By simplifying workflows and mitigating process risks, the Cellvento® ModiFeed platform empowers researchers and developers to enhance their approach to glycosylation modulation, ultimately contributing to improved therapeutic outcomes and patient care.
Modulating and Targeting Galactosylation Profiles of Monoclonal Antibodies
Galactosylation is the attachment of galactose monosaccharides to a glycoprotein side chain and is a key determinant for parameters such as serum half-life and biological activity. For biosimilars, optimizing titer while controlling galactosylation levels is critical to match the performance of the reference molecule.
Cellvento® ModiFeed Gal+ & Gal- Feeds for Tailored Glycosylation in CHO Cell Culture
The Cellvento® ModiFeed Gal+ and Cellvento® ModiFeed Gal- products were specifically developed to optimize and control terminal galactosylation in any Chinese hamster ovary (CHO) cell fed-batch system. These innovative products can be used in two ways:
- Use as stand-alone feed for maximum glycosylation shift. Feed 100% Cellvento® ModiFeed Gal+ or Cellvento® ModiFeed Gal- throughout the duration of a fed-batch process to increase or decrease galactosylation levels, respectively. Cellvento® ModiFeed Gal+ Feed increases terminal galactosylation (G1/G2(F)) by increasing the concentration of specific modulating raw materials. In contrast, Cellvento® ModiFeed Gal- Feed increases G0(F) and decreases G1/G2(F).
- Feeding Cellvento® ModiFeed Gal+ or Cellvento® ModiFeed Gal- feed in combination with Cellvento® ModiFeed Prime feed can create a titratable response, allowing galactosylation to be tailored to a desired level.
Case Study: Effect of a Tailored Feeding Strategy on the mAb Glycosylation Profile in Various Media and CHO Cell Clones
This case study describes the use of feeding different ratios of the feeds Cellvento® ModiFeed Prime/Gal+ or Cellvento® ModiFeed Prime/Gal- to demonstrate a titratable effect for galactosylation modulation and the potential for a profile shift. All conditions were fed 17.5% v/v feed throughout the 14-day fed batch.
This case study describes the use of the Cellvento® ModiFeed toolkit utilizing Cellvento® ModiFeed Prime feed as a foundation, incorporating raw materials that can influence glycosylation on glycoproteins. The goal is to enable the user to customize glycosylation profiles and be capable of targeting a desired glycosylation profile.
The impact of Cellvento® ModiFeed Gal+ and Gal- feeds on protein galactosylation is shown in Figure 2. The baseline glycosylation profile of two clones using different basal medium fed Cellvento® ModiFeed Prime is shown, along with the ability of Cellvento® ModiFeed Gal+ to increase terminal galactosylation (Gal) and Cellvento® ModiFeed Gal- to increase terminal N-Acetylglucosamine (GlcNAc). High mannose glycan (Man5) shown if detected.
Figure 2.Glycoform composition using galactosylation modulating feeds (Gal+, Gal-) compared to baseline Cellvento® ModiFeed Prime feed (Prime).
(Data on day 14 for two different clones in two different media.)
Figure 3 illustrates how combinations of Cellvento® ModiFeed Gal+ or Cellvento® ModiFeed Gal- Feed with Cellvento® ModiFeed Prime can be used to titrate galactosylation levels or to achieve a specific target galactosylation profile.
Figure 3A shows the glycosylation profile of a mAb produced from a CHOZN® GS cell line on day 14 using Cellvento® 4CHO medium. A higher percentage of Cellvento® ModiFeed Gal+ in the feed mix, which translates to a higher concentration of galactose increasing modulators, led to more galactosylated glycoforms G1/G2(F) while the titer remained constant.
Similarly, Cellvento® ModiFeed Gal- feed can be mixed with Cellvento® ModiFeed Prime feed to create a titratable response to deliver lower galactosylation levels. Figure 3B shows the glycosylation profile of a CHOZN® GS cell line on day 14 grown in EX-CELL® Advanced medium. Higher percentages of Cellvento® ModiFeed Gal- COMP in the feed mix led to less galactosylated glycoforms G1/G2(F). Titer/productivity was confirmed to be constant regardless of the feed mix.
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Figure 3. Combinations of Cellvento® ModiFeed Prime feed with either Cellvento® ModiFeed Gal+ (A) or Cellvento® ModiFeed Gal- feed (B) can be used to achieve different galactosylation levels by altering the ratio of the combined feed.
Tuning the Sialylation Level – A Titratable Process
Sialic acid is found as the final sugar unit in the glycosylation chains, either N- or O-linked to numerous glycoproteins. Specifically, the sialylation of the N-glycosylated Fc region of IgG has been shown to have substantial anti-inflammatory properties. Changes in the level of sialylation are also factors that shape the development of certain autoimmune diseases. Depending on the molecule and the application, terminal sialic acid may reduce the rate of clearance, or can increase the anti-inflammatory function of the recombinant protein. As a result, high sialylation levels can help to increase stability, safety, and efficacy of the protein of interest. At the same time, full control of the sialylation levels is critical.
Cellvento® ModiFeed Sial+ Feed for Efficient Sialylation Control in CHO Cell Culture
The Cellvento® ModiFeed Sial+ product was specifically developed to increase terminal sialylation in any CHO fed-batch system. This innovative product can be used in two ways:
- As a stand-alone feed for maximum sialylation in which 100% Cellvento® ModiFeed Sial+ is fed throughout the duration of a fed-batch process to maximize sialylation levels.
- Feeding Cellvento® ModiFeed Sial+ feed in combination with Cellvento® ModiFeed Prime feed can be used to increase terminal sialic acid to a desired level.
Case Study: Effect of a Tailored Feeding Strategy on Sialylation Profile and Glycoform Composition
This case study describes how different feeding ratios of the feeds Cellvento® ModiFeed Prime/Sial+ create a titratable effect for sialylation modulation and highlights glycoform distribution and compositions.
Cellvento® ModiFeed Sial+ feed impacts sialylation on proteins of interest including hormones, Fc-Fusion proteins, and other highly sialylated proteins. Figure 4 shows the baseline glycosylation profile of two clones using different basal media fed Cellvento® ModiFeed Prime feed compared to feeding Cellvento® ModiFeed Sial+ and its ability to increase terminal sialylation of N-linked glycans.
Figure 4.Glycoform composition comparing the use of sialylation modulating feed Cellvento® ModiFeed Sial+ feed (Sial+) compared to baseline Cellvento® ModiFeed Prime feed (Prime).
(Data on day 14 for two different clones in two different media.)
Cellvento® ModiFeed Sial+ feed should be used at 100% to maximize sialylation on terminal glycans. However, it can be mixed with Cellvento® ModiFeed Prime feed to increase terminal sialic acid to a desired target level. Figure 5A shows the sum of all glycan species according to their terminal glycosylation; a higher percentage of Cellvento® ModiFeed Sial+ in the feed mix increased sialylation level and reduced the concentration of other glycoforms of a fusion protein on day 12. Figure 5B demonstrates the influence of the feed composition on the sialic acid glycoform composition.
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Figure 5.Titration of the sialylation profile of CHO cell culture by altering the ratio of the Cellvento® ModiFeed Prime and Cellvento® ModiFeed Sial+ feed combination: Effect on A) Glycoform composition and B) sialylated species .
(Pink diamonds represent sialic acid, yellow circles galactose, blue squares GlcNAc, green circles mannose, and red triangles fucose; day 12 values)
Figure 6 shows the effect of Cellvento® ModiFeed Sial+ on a different CHOZN® clone producing a molecule with more complex glycans. The tetra-sialylated glycans were increased on day 14 for this molecule; O-glycans are also increased by Cellvento® ModiFeed Sial+ (data not shown).
Figure 6.Titration of the sialylation profile of CHO cell culture using a combination of Cellvento® ModiFeed Prime and Cellvento® ModiFeed Sial+ feeds, showing the effect of altering the ratio of the combined feed on sialylated species (graph shows day 14 values).
Advantages of a Tailored Feeding Strategy
Development of upstream processes necessitates a meticulous orchestration of medium and feed formulations to optimize culture conditions and achieve the desired glycosylation profile. Given the critical role of glycosylation as an important quality attribute for glycoproteins, the Cellvento® ModiFeed platform is a valuable tool for both biologic innovators and biosimilar manufacturers seeking to control protein glycosylation.
Beyond its robust performance, the Cellvento® ModiFeed platform offers a streamlined workflow and easier handling as a compacted, highly concentrated, single-part, pH-neutral feed format. The products are stable when hydrated which ensures storage flexibility and minimizes contamination risks, thereby enhancing operational efficiency.
As described above, the right combination of medium and feed, along with precise control of culture conditions, is integral to achieving optimal glycosylation profiles. By empowering researchers and developers to modulate glycosylation with precision, the Cellvento® ModiFeed platform significantly contributes to improved therapeutic outcomes.
In addition to our catalogue media and feeds, we also offer custom cell culture media, feeds, supplements, buffers, and concentrates. Learn more: Custom Solutions for Cell Culture
Comprehensive documentation tailored to specific cell culture needs is provided with all Cellvento® ModiFeed platform products. Learn more: Emprove® Dossiers for Cell Culture Media in Upstream Processes
For more information and data on tailored feeding strategy, watch our webinar:
“Tailoring Product Critical Quality Attributes with a Novel Feed Tool Kit”
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