Controlling glycation of recombinant antibody in fed‐batch cell cultures

Abstract

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.

In vitro and in vivo modifications of recombinant and human IgG antibodies

“…136 Glycation can be controlled by optimizing cell culture conditions. 141 A mAb containing 17% glycation did not show any structural difference compared with the main peak with no glycation.…”

5.3 Nutrient Media for Cell Culture Technology

“…A large number of studies have used glucose concentration as the control criterion for the development of fed-batch cultures [59,60,54,53,61,62]. They investigated the reduction of the lactate production using a glucose feed that is maintained at about 0.3 to 1.5 mmol L -1 in the bioreactor [60,54], initiating a metabolic shift towards a more efficient glucose utilisation (see Figure 5.3.1).…”

“…They investigated the reduction of the lactate production using a glucose feed that is maintained at about 0.3 to 1.5 mmol L -1 in the bioreactor [60,54], initiating a metabolic shift towards a more efficient glucose utilisation (see Figure 5.3.1). Additionally, other studies have investigated how the glucose limitation in fed-batch culture can affect the degree of glycosylation [61] and glycation [62] of recombinant proteins. These studies have shown that, although a reduction in the availability of glucose as a sugar reducer in the culture medium is capable of reducing the glycation of the protein of interest, very low glucose levels may decrease sialylation and affect the extension of N-glycan chains of the recombinant protein, which can be detrimental to its biological quality.…”

Glycation of antibodies: Modification, methods and potential effects on biological functions

“…5 The temperature, pH, time, and ionic strength, which are kept close to physiological conditions, can affect the kinetics and extent of glycation. 30 The types of sugars present, such as hexose sugars, for example, and the specific reactivity of the accessible amino groups affect the protein glycation, and therefore increase the heterogeneity of therapeutic proteins. 31 During storage of therapeutic mAbs, glycation can be introduced by reducing sugars in the formulation.…”