Cell culture process metabolomics together with multivariate data analysis tools opens new routes for bioprocess development and glycosylation prediction.

Multivariate latent variable methods have become a popular and versatile toolset to analyze bioprocess data in industry and academia. This work spans such applications from the evaluation of the role of the standard process variables and metabolites to the metabolomics level, that is, to the extensive number metabolic compounds detectable in the extracellular and intracellular domains. Given the substantial effort currently required for the measurement of the latter groups, a tailored methodology is presented that is capable of providing valuable process insights as well as predicting the glycosylation profile based on only four experiments measured over 12 cell culture days. An important result of the work is the possibility to accurately predict many of the glycan variables based on the information of three experiments. An additional finding is that such predictive models can be generated from the more accessible process and extracellular information only, that is, without including the more experimentally cumbersome intracellular data. With regards to the incorporation of omics data in the standard process analytics framework in the future, this works provides a comprehensive data analysis pathway which can efficiently support numerous bioprocessing tasks.

Mechanistic reconstruction of glycoprotein secretion through monitoring of intracellular N-glycan processing.

N-linked glycosylation plays a fundamental role in determining the thermodynamic stability of proteins and is involved in multiple key biological processes. The mechanistic understanding of the intracellular machinery responsible for the stepwise biosynthesis of N-glycans is still incomplete due to limited understanding of in vivo kinetics of N-glycan processing along the secretory pathway. We present a glycoproteomics approach to monitor the processing of site-specific N-glycans in CHO cells. On the basis of a model-based analysis of structure-specific turnover rates, we provide a kinetic description of intracellular N-glycan processing along the entire secretory pathway. This approach refines and further extends the current knowledge on N-glycans biosynthesis and provides a basis to quantify alterations in the glycoprotein processing machinery.

Generation of site-distinct N-glycan variants for in vitro bioactivity testing.

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

Glycosylation flux analysis reveals dynamic changes of intracellular glycosylation flux distribution in Chinese hamster ovary fed-batch cultures.

N-linked glycosylation of proteins has both functional and structural significance. Importantly, the glycan structure of a therapeutic protein influences its efficacy, pharmacokinetics, pharmacodynamics and immunogenicity. In this work, we developed glycosylation flux analysis (GFA) for predicting intracellular production and consumption rates (fluxes) of glycoforms, and applied this analysis to CHO fed-batch immunoglobulin G (IgG) production using two different media compositions, with and without additional manganese feeding. The GFA is based on a constraint-based modeling of the glycosylation network, employing a pseudo steady state assumption. While the glycosylation fluxes in the network are balanced at each time point, the GFA allows the fluxes to vary with time by way of two scaling factors: (1) an enzyme-specific factor that captures the temporal changes among glycosylation reactions catalysed by the same enzyme, and (2) the cell specific productivity factor that accounts for the dynamic changes in the IgG production rate. The GFA of the CHO fed-batch cultivations showed that regardless of the media composition, galactosylation fluxes decreased with the cultivation time more significantly than the other glycosylation reactions. Furthermore, the GFA showed that the addition of Mn, a cofactor of galactosyltransferase, has the effect of increasing the galactosylation fluxes but only during the beginning of the cultivation period. The results thus demonstrated the power of the GFA in delineating the dynamic alterations of the glycosylation fluxes by local (enzyme-specific) and global (cell specific productivity) factors.

Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation.

Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.

Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality.

Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448-1458. © 2017 Wiley Periodicals, Inc.

Robust factor selection in early cell culture process development for the production of a biosimilar monoclonal antibody.

This work presents a multivariate methodology combining principal component analysis, the Mahalanobis distance and decision trees for the selection of process factors and their levels in early process development of generic molecules. It is applied to a high throughput study testing more than 200 conditions for the production of a biosimilar monoclonal antibody at microliter scale. The methodology provides the most important selection criteria for the process design in order to improve product quality towards the quality attributes of the originator molecule. Robustness of the selections is ensured by cross-validation of each analysis step. The concluded selections are then successfully validated with an external data set. Finally, the results are compared to those obtained with a widely used software revealing similarities and clear advantages of the presented methodology. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:181-191, 2017.

Screening and assessment of performance and molecule quality attributes of industrial cell lines across different fed-batch systems.

The major challenge in the selection process of recombinant cell lines for the production of biologics is the choice, early in development, of a clonal cell line presenting a high productivity and optimal cell growth. Most importantly, the selected candidate needs to generate a product quality profile which is adequate with respect to safety and efficacy and which is preserved across cell culture scales. We developed a high-throughput screening and selection strategy of recombinant cell lines, based on their productivity in shaking 96-deepwell plates operated in fed-batch mode, which enables the identification of cell lines maintaining their high productivity at larger scales. Twelve recombinant cell lines expressing the same antibody with different productivities were selected out of 470 clonal cell lines in 96-deepwell plate fed-batch culture. They were tested under the same conditions in 50 mL vented shake tubes, microscale and lab-scale bioreactors in order to confirm the maintenance of their performance at larger scales. The use of a feeding protocol and culture conditions which are essentially the same across the different scales was essential to maintain productivity and product quality profiles across scales. Compared to currently used approaches, this strategy has the advantage of speeding up the selection process and increases the number of screened clones for getting high-producing recombinant cell lines at manufacturing scale with the desired performance and quality.

Tailoring recombinant protein quality by rational media design.

Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function.