Comparative glycomics analysis of influenza Hemagglutinin (H5N1) produced in vaccine relevant cell platforms

Abstract

Hemagglutinin (HA) is the major antigen in influenza vaccines, and glycosylation is known to influence its antigenicity. Embryonated hen eggs are traditionally used for influenza vaccine production, but vaccines produced in mammalian and insect cells were recently licensed. This raises the concern that vaccines produced with different cell systems might not be equivalent due to differences in their glycosylation patterns. Thus, we developed an analytical method to monitor vaccine glycosylation through a combination of nanoLC/MS(E) and quantitative MALDI-TOF MS permethylation profiling. We then used this method to examine glycosylation of HAs from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five, expresSF+ and glycoengineered expresSF+), and a human cell line (HEK293). Our results demonstrated that (1) sequon utilization is not necessarily equivalent in different cell types, (2) there are quantitative and qualitative differences in the overall N-glycosylation patterns and structures produced by different cell types, (3) ∼20% of the N-glycans on the HAs produced by High Five cells are core α1,3-fucosylated structures, which may be allergenic in humans, and (4) our method can be used to monitor differences in glycosylation during the cellular glycoengineering stages of vaccine development.

Figure 1

Work flow diagram of the glycoprotein analysis approach used in this study. Branches indicate intact glycopeptide analysis by nanoLC/MS^E and glycan permethylation profiling.

Figure 2

Stacked plot of glycosylation site 4. An example is shown for each cell platform. The colors in the spectrum represent: red, y ions; blue, b ions; green, y/b ions after neutral loss; grey-unassigned ions by Biopharmalynx (some have been manually assigned as indicated). Monosaccharide symbols: , GlcNAc; , Man; , Fuc. Oxonium ions at 204.09 and 528.19 are indicated.

Figure 3

MS^E spectrum of High Five™ derived HA glycopeptide (Hex₃dHex₂HexNAc₂)DQICIGYHANNSTEQVDTIMEK containing site one. The colors in the spectrum represent: red, y ions; blue, b ions; green, y/b ions after neutral loss; grey-unassigned ions by Biopharmalynx (some have been manually assigned as indicated). Monosaccharide symbols: , GlcNAc; , Man; , Fuc. Oxonium ions at 204.09 and 528.19 are indicated.

Figure 4

MS^E spectrum of HEK293 derived HA glycopeptide (NeuAc₂dHex₁Hex₄HexNAc₅)-SYNNTNQEDLLVLWGIHHPNDAAEQTK, m/z [M+H]⁺ 5499.33, containing site 4. The colors in the figure indicate the following: red, y ions; blue, b ions; green, y/b ions after neutral loss; pink, glycopeptides after neutral loss assigned by Biopharmalynx; grey-unassigned ions by Biopharmalynx (some have been manually assigned as indicated). Monosaccharide symbols: , GlcNAc; , Man; , Fuc; , GalNAc; , Gal; , NeuAc. Oxonium ions at 274.09 and 495.18 are indicated.

Figure 5

MS^E spectrum of SfSWT-7 derived HA glycopeptide, m/z 3559.61 [M+H]+ (Hex₄dHex₁HexNAc₃)LYQNPTTYISVGTSTLNQR, containing site 4. The colors in the figure indicate the following: red, y ions; blue, b ions; green, y/b ions after neutral loss; pink- glycopeptides after neutral loss assigned by Biopharmalynx; gray; unassigned ions by the Biopharmalynx (some have been manually assigned as indicated). Monosaccharide symbols: , GlcNAc; , Man, Fuc. Peptide fragments with glycosyl neutral loss are indicated.

Figure 6

3-D structure of HA A/Viet Nam/1203/2004 monomer analyzed in this study (PDB: 2IBX). All glycosylation sites are highlighted in yellow. Partial glycan structures observed in the X-ray structure are shown. The glycosylation site number is noted in parentheses. The glycosylation site ASN number and position is indicated. Protein data from the NCBI structural database were processed with CN3D 1.0 software. In this study glycosylation sites at ASN 209 and 302 were only occupied in insect cell derived HA and are therefore cell type dependant.

Figure 7

The SfSWT-7 cell derived HA has glycan compositions that are more mammalian like those of its parent expresSF+ cells. Comparison of egg, expresSF+, and SfSWT-7 derived HA released and permethylated glycans as analyzed by MALDI-TOF MS are shown. Measurements were performed in triplicate and subclass individual isobars were summed. Error bars represent one standard deviation. Example compositions of each class are shown above histogram bars. The SfSWT-7 mammalian like complex glycan subclass was approximately one third that of egg whereas parent expresSF+ produced HA had no mammalian like complex glycans.