Haemagglutinin quantification and identification of influenza A&B strains propagated in PER.C6 cells: a novel RP-HPLC method.

The major antigenic determinant of influenza A and B virus is haemagglutinin (HA). The HA content is an important specification of influenza vaccines. HA in vaccines has typically been quantified by single-radial-immunodiffusion (SRID). However, SRID is a laborious and low throughput assay. Moreover, sensitivity, accuracy, and precision, especially for non-purified (in-process) influenza virus is relatively low. We present a novel method for quantification of HA in influenza viral cultures as well as for the identification of HA from individual influenza strains in trivalent vaccines. The method is based on the separation of HA(1), the hydrophilic subunit of HA, from the more hydrophobic viral and matrix components by reversed-phase high performance liquid chromatography (RP-HPLC). The HA(1) peak area is demonstrated to be proportional to the level of HA in non-purified, semi-purified and purified vaccine products of various epidemic and pandemic influenza A and B strains propagated in PER.C6((R)) cell cultures. The RP-HPLC assay selectivity allows for the simultaneous identification and quantification of HA(1) from influenza A and B strains in the yearly revised trivalent vaccines for epidemic outbreaks.

High-level expression of recombinant IgG in the human cell line per.c6.

The number of therapeutic monoclonal antibodies in production is expected to rise rapidly in the next few years. As a result, there is much focus on the optimization of antibody expression platforms. Several issues are important including the speed of transition from bench to manufacturing, yield of IgG, and quality (particularly of the glycan structures present on immunoglobulins). We have characterized the human cell line PER.C6 for its ability to produce recombinant IgG. Production yields are still being optimized, but in nonfed batch culture, PER.C6 is able to grow to a cell density of 5 x 10(6) cells/mL and produce 300-500 mg/L IgG; this is likely to increase significantly in fed batch cultures. The generation of antibody-producing cell lines is fast, as rounds of amplification of inserted genes are not required for high production yields. The gene copy number of inserted genes is in the region of 1-10 copies per genome. In addition, PER.C6 is a human cell line, and so does not add glycans, which are immunogenic in humans. A core fucose molecule is essentially always present, and galactose residues are present at a physiological level (0, 1, and 2 galactose residues per glycan are present at a ratio of 1:2:1). No hybrid or high-mannose structures are seen.

On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins.

An on-line high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is described for the rapid characterization of any type of oligosaccharide released from glycoproteins. The procedure can be applied without further manipulation to fractions collected from a high-performance anion-exchange chromatography-pulse amperometric detection (HPAEC-PAD) system commonly used for glycosylation mapping of glycoproteins, or to a pool of oligosaccharides directly released from glycoproteins. The system consists of a porous graphitized high-performance chromatography column (Hypercarb) coupled to a quadrupole time-of-flight (TOF) mass spectrometer. Oligosaccharides are eluted from the column with a gradient of ammonium acetate/acetonitrile and directly identified following in-source fragmentation. Some applications of the method are presented, as well as information about the spectra and fragmentation behavior observed for N- and O-linked oligosaccharides released from some recombinant glycoproteins. Low femtomole limits of detection are achieved using proper miniaturization.