Dana C. Andersen

The oligosaccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties.

In this review, we organize the recent data concerning the effects of bioprocess factors on the oligosaccharide structure of human therapeutic glycoproteins, with particular emphasis on the influence of the host cell. We also discuss the effect of oligosaccharide structure on glycoprotein properties, including antigenicity, immunogenicity and plasma clearance rate.

Recombinant protein expression for therapeutic applications

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Many of these applications involve complex glycoproteins and antibodies with relatively high production needs. These demands have driven the development of a variety of improvements in protein expression technology, particularly involving mammalian and microbial culture systems.

Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator

Human tissue-type plasminogen activator (t-PA) contains a variably occupied glycosylation site at Asn-184 in naturally produced t-PA and in t-PA produced in recombinant Chinese hamster ovary (CHO) cells. The presence of an oligosaccharide at this site has previously been shown to reduce specific activity and fibrin binding. In this report, the site occupancy of t-PA is shown to increase gradually over the course of batch and fed-batch CHO cultures. Additional cell culture factors, including butyrate and temperature, are also shown to influence the degree of glycosylation. In each of these cases, conditions with decreased growth rate correlate with increased site occupancy. Investigations using quinidine and thymidine to manipulate the cell cycle distribution of cultures further support this correlation between site occupancy and growth state. Comparison of the cell cycle distribution across the range of cell culture factors investigated shows a consistent relationship between site occupancy and the fraction of cells in the G(0)/G(1) phase of the cell cycle. These results support a correlation between growth state and site occupancy, which fundamentally differs from site occupancy trends previously observed and illustrates the importance of the growth profile of CHO cultures in producing consistently glycosylated recombinant glycoproteins.

Controlling glycation of recombinant antibody in fed‐batch cell cultures

Protein glycation is a non‐enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in‐house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post‐translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose—the only source of reducing sugar in our fed‐batch cultures—would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2‐L and 400‐L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second‐order chemical reaction that is first order with respect to both glucose and non‐glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture. Biotechnol. Bioeng. 2011;108: 2600–2610.

Analyzing mixtures of amino acids and carbohydrates using bi-modal integrated amperometric detection

Described in this work is a new detection methodology - bi-modal integrated amperometric detection - for identifying peaks and as a tool for solving difficult separation problems. Bi-modal integrated amperometry makes it possible to selectively detect amino acids, amino sugars, and carbohydrates following their separation by anion-exchange. Selectivity is gained by two different methods of integrating anodic current on an otherwise identical waveform. As with the single-mode integrated amperometry reported previously, the limits of detection are in the femtomole range and linear calibration plots are possible over three orders of magnitude. This new detection method does not require analyte derivatization. The practical utility of this new technique is demonstrated in the analysis of amino acids and sugars in a recombinant mammalian cell culture medium.

Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation

ABSTRACT For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal (“afucosylation”). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)‐enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND‐enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies—mAb1, mAb2, and mAb3—at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND‐enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development.