Søren Kofoed Rasmussen

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

Fed‐batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N‐glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation‐related product quality. In this work, different fed‐batch processes with two chemically defined proprietary media and feeds were studied using two IgG‐producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N‐glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by‐products such as NH4+ and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case‐by‐case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP‐GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α‐1,3‐mannosyl‐glycoprotein 2‐β‐N‐acetylglucosaminyltransferase (GnTI) and UDP‐GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP‐Gal biosynthesis, which was observed to be both cell line and cultivation condition‐dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP‐Gal availability. All these findings are important for optimization of fed‐batch culture for improving IgG production and directing glycosylation quality. Biotechnol. Bioeng. 2015;112: 521–535.

Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use.

The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well‐characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost‐efficient cell banking and single‐batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled‐up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic‐, protein‐, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition. Biotechnol. Bioeng. 2011;108:2171–2181.

Recombinant antibody mixtures: production strategies and cost considerations.

Recombinant monoclonal antibodies have during the last two decades emerged as a very successful class of biological drugs for the treatment of a variety of different diseases used either as biological mono therapy or in combination with small molecule based drugs. Recombinant antibody mixtures offering targeting of more than one antigen is one of the new promising antibody technologies resulting in higher therapeutic effectiveness and/or broader reactivity. Such recombinant antibody mixtures can in principle be manufactured by different approaches but two main strategies is often applied, either individual manufacturing of the constituent antibodies or single batch manufacturing of the recombinant antibody mixture. Symphogen has developed an expression platform, Sympress™, allowing single batch manufacturing of recombinant antibody mixtures, while other companies are currently using a manufacturing strategy based on production of the individual constituent monoclonal antibodies. An overview and comparison of the different approaches with focus on the challenges in terms of cell banking strategy, manufacturing approach, and strategies for release and characterization will be reviewed in the present manuscript. Furthermore, the two manufacturing approaches are compared based on different parameters such as development timelines, preclinical developmental costs, and manufacturing cost of goods sold (COGS). We conclude that the single batch manufacturing approach expressing a mixture of full length IgG provides a robust and reproducible platform that can be used for cost effective manufacturing of recombinant antibody mixtures.

Manufacture of recombinant polyclonal antibodies

Polyclonal antibody therapy in the form of hyper-immune serum has for more than a century been used for treatment of many infectious diseases. However, with the emergence of first antibiotics and later recombinant monoclonal antibody therapy, the use of hyper-immune serum has declined. The main reason for this is that methods for consistent manufacturing of safe hyper immune immunoglobulin products have been lacking. In contrast, manufacturing processes of recombinant monoclonal antibodies follow a well established schedule and it appears obvious to use similar methods to produce recombinant polyclonal products. However, the methods for monoclonal antibody manufacturing are, for several reasons, not directly applicable to generation and manufacture of polyclonal recombinant antibodies. A new production strategy based on recombinant mammalian producer cells has recently been developed to support consistent generation of recombinant polyclonal antibodies for therapeutic use. This review describes aspects of this novel technology with emphasis on the generation, production and characterization procedures employed, and provides comparison with alternative polyclonal and monoclonal antibody manufacturing strategies.

Capturing the Natural Diversity of the Human Antibody Response against Vaccinia Virus

ABSTRACT The eradication of smallpox (variola) and the subsequent cessation of routine vaccination have left modern society vulnerable to bioterrorism employing this devastating contagious disease. The existing, licensed vaccines based on live vaccinia virus (VACV) are contraindicated for a substantial number of people, and prophylactic vaccination of large populations is not reasonable when there is little risk of exposure. Consequently, there is an emerging need to develop efficient and safe therapeutics to be used shortly before or after exposure, either alone or in combination with vaccination. We have characterized the human antibody response to smallpox vaccine (VACV Lister) in immunized volunteers and isolated a large number of VACV-specific antibodies that recognize a variety of different VACV antigens. Using this broad antibody panel, we have generated a fully human, recombinant analogue to plasma-derived vaccinia immunoglobulin (VIG), which mirrors the diversity and specificity of the human antibody immune response and offers the advantage of unlimited supply and reproducible specificity and activity. The recombinant VIG was found to display a high specific binding activity toward VACV antigens, potent in vitro VACV neutralizing activity, and a highly protective efficacy against VACV challenge in the mouse tail lesion model when given either prophylactically or therapeutically. Altogether, the results suggest that this compound has the potential to be used as an effective postexposure prophylaxis or treatment of disease caused by orthopoxviruses.

Single-Batch Production of Recombinant Human Polyclonal Antibodies

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the SympressTM I technology. The SympressTM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the SympressTM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the SympressTM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the SympressTM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.

A multi‐pronged investigation into the effect of glucose starvation and culture duration on fed‐batch CHO cell culture

In this study, omics‐based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed‐batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity and quality. Titer and N‐glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N‐glycosylation of mAbs was dependent on the degree of starvation during early stationary phase of the fed‐batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP‐GlcNAc and UDP‐GalNAc, but increased the levels of UDP‐Glc and UDP‐Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early stationary phase. These analyses offered a systematic view of the intrinsic properties of these cells and allowed us to explore the root causes correlating culture duration with variations in the productivity and glycosylation quality of monoclonal antibodies produced with CHO cells. Biotechnol. Bioeng. 2015;112: 2172–2184.

Recombinant antibody mixtures; optimization of cell line generation and single-batch manufacturing processes

Background Recombinant antibody mixtures represent an important new class of antibody therapeutics as demonstrated by the increasing amount of literature showing that combinations of two or more antibodies show superiority compared to monoclonal antibodies (mAbs) for treatment of cancer and infectious diseases [1-5]. Sym004, composed of two antibodies targeting non-overlapping epitopes of the epidermal growth factor receptor (EGFR) act in a synergistic manner to induce an efficient internalization of EGFR leading to subsequent degradation and exhibit superior anticancer efficacy as demonstrated in several preclinical in vivo models [5]. At Symphogen A/S, we have developed an expression platform, SympressTM, for cost-efficient production of antibody mixtures. The antibody mixtures are produced using a single-batch manufacturing approach where a polyclonal working cell bank (pWCB) prepared by mixing the individual stable cell lines producing all the desired antibodies is used as seed material for a bioreactor process [6]. By using a single-batch approach the CMC development costs of antibody mixtures are comparable to costs for monoclonal antibodies. However, the single-batch manufacturing approach raises questions with regard to control of composition ratios, compositional stability and robustness of the cell banking procedure. Here, we present experimental data addressing these key questions and demonstrate that mixtures of recombinant antibodies can be produced under predictable, reproducible and stable conditions using the SympressTM technology.