The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.

During the manufacturing of protein biologics, product variability during cell culture production and harvest needs to be actively controlled and monitored to maintain acceptable product quality. To a large degree, variants that have previously been described are covalent in nature and are easily analyzed by a variety of techniques. Here, we describe a noncovalent post translational modification of recombinantly expressed antibodies, containing variable domain tryptophans, that are exposed to culture media components and ambient laboratory light. The modified species, designated as conformer, can be monitored by hydrophobic interaction chromatography and often exhibits reduced potency. We studied conformer formation and identified key elements driving its accelerated growth using an IgG2 monoclonal antibody. Conformer is a result of a noncovalent interaction of the antibody with riboflavin, an essential vitamin added to many production cell culture formulations. Chemical and physical factors that influence the impact of riboflavin are identified, and methods for process control of this product quality attribute are addressed in order to prevent loss of antibody potency and potential safety issues. Identifying therapeutic antibody drug candidates with the potential to form conformers can be performed early in development to avoid this undesirable product quality propensity.