Size distribution analysis of influenza virus particles using size exclusion chromatography

Abstract

Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity.

Interference chromatography: a novel approach to optimizing chromatographic selectivity and separation performance for virus purification

“…Therefore, the ability of this new technology chromatography membrane to bind virus at resin capacity at membrane flow rates and using interference multivalent ions was evaluated and exploited for a one-step NDV purification and at constant pH of 8.2. The interference chromatography approach creates interference in the sample (for this study, it the interactions were mainly charge shielding and van der Waals forces, similar to methodologies used previously [52,53] and the equilibration buffer in order to influence separation between biomolecules in the matrix, i.e. product-related and process-related molecules.…”

“…This interference method allows for improvements in performance of any chromatographic approach, such as superior resolution, improved binding capacity or augmented flow through productivity. Variations of this technique with anion exchange resins or membranes have been successfully used in the past for virus purification of Influenza H1N1 virus [52] and Influenza A/B virus [53]. One study showed that anions from triprotic kosmostropes, phosphate and citrate, with anion-exchange resins having quaternary amine or primary amine functional groups showed good virus binding, with improved binding by the salt-tolerant primary amine in phosphate buffer at Fig.…”

“…Proteins were analyzed in triplicate, virus samples (n = 3) were pooled and titered. Means ±SD are shown pH 8.0 [52]. The other study used a continuous twocolumn approach with the first column of big bead AEX resin and the second one with a primary amine membrane adsorber with multivalent ions and both chromatography steps run in the negative-binding mode (also known as Flow Through Chromatography) to purify Influenza virus [53].…”

“…Depending on the chromatographic mode, the target molecule can flow through the media while impurities bind (negative mode [53];) or the target molecule is bound to the chromatographic support and the impurities pass through. The latter option, often referred to as bind-and-elute mode ( [52]; and the current study), then requires an elution step to remove the target molecule from the solid phase. In this study with the binding of NDV to the HD-Q hydrogel matrix, the host cell proteins flowed through the chromatography membrane and then the NDV was reverse eluted in strong salt (1 M NaCl) conditions to recover the virus.…”

Characterization and stabilization in process development and product formulation for super large proteinaceous particles

“…However, as the development of SEC technology, commercial analytical columns with pore size of matrix above 100 nm are available now, making it possible to analyze most large virus and VLPs as well as aggregates in the range of 10–100 nm [25]. The TSKgel G6000PWxl column, whose matrix diameter was determined from 20 nm to 2000 nm and the mean value of 250 nm, was reported to separate influenza A virus particle monomers, aggregates and fragments [26].…”