Isolation by multistep chromatography improves the consistency of secreted recombinant influenza neuraminidase antigens

Abstract

Recombinant protein-based approaches are ideally suited for producing vaccine antigens that are not overly abundant in viruses, such as influenza neuraminidase (NA). However, obtaining sufficient quantities of recombinant viral surface antigens remains challenging, often resulting in the use of chimeric proteins with affinity tags that can invariably impact the antigen’s properties. Here, we developed multistep chromatography approaches for purifying secreted recombinant NA (rNA) antigens that are derived from recent H1N1 and H3N2 viruses and produced using insect cells. Analytical analyses showed that these isolation procedures yielded homogenous tetrameric rNA preparations with consistent specific activities that were not possible from a common immobilized metal affinity chromatography purification procedure. The use of classical chromatography improved the rNA tetramer homogeneity by removing the requirement of the N-terminal poly-histidine affinity tag that was shown to promote higher order rNA oligomer formation. In addition, these procedures reduced the specific activity variation by eliminating the exposure to Ni2+ ions and imidazole, with the latter showing pH and NA subtype dependent effects. Together, these results demonstrate that purification by multistep chromatography improves the homogeneity of secreted rNAs and eliminates the need for affinity tag-based approaches that can potentially alter the properties of these recombinant antigens.