# Variation of Site-Specific Glycosylation Profiles of Recombinant Influenza Glycoproteins

## TL;DR

This study examines how the glycosylation patterns of influenza proteins vary based on factors like strain and production methods.

## Contribution

The paper introduces a method to quantify glycosylation variation using spectral similarity scores and identifies key factors influencing it.

## Key findings

- Strain differences caused the most glycosylation variation (similarity score = 383), while replicates showed minimal variation (similarity score = 957).
- Four distinct glycosylation classes were identified for most sites across the analyzed proteins.
- Only one of two adjacent glycosylation sites in an HA variant was occupied, revealed by rare proteases.

## Abstract

This work presents a detailed determination of site-specific N-glycan distributions of the recombinant influenza glycoproteins hemagglutinin (HA) and neuraminidase. Variation in glycosylation among recombinant glycoproteins is not predictable and can depend on details of the biomanufacturing process as well as details of protein structure. In this study, recombinant influenza proteins were analyzed from eight strains of four different suppliers. These include five HA and three neuraminidase proteins, each produced from a HEK293 cell line. Digestion was conducted using a series of complex multienzymatic methods designed to isolate glycopeptides containing single N-glycosylated sites. Site-specific glycosylation profiles of intact glycopeptides were produced using a recently developed method and comparisons were made using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 999), whereas digestion replicates and injection replicates showed relatively little variation (similarity score = 957). Notably, glycan distributions for homologous regions of influenza glycoprotein variants showed low variability. Due to the multiple possible sources of variation and inherent analytical difficulties in site-specific glycan determinations, variations were individually examined for multiple factors, including differences in supplier, production batch, protease digestion, and replicate measurement. After comparing all glycosylation distributions, four distinguishable classes could be identified for the majority of sites. Finally, attempts to identify glycosylation distributions on adjacent potential N-glycosylated sites of one HA variant were made. Only the second site (NnST) was found to be occupied using two rarely used proteases in proteomics, subtilisin and esperase, both of which did selectively cleave these adjacent sites.

•Variation of site-specific glycosylation measurements is not well understood.•Glycopeptide abundance distribution spectra can quantitatively describe variation.•Factors that produce the least variation include replicates and batches.•Factors that produce the most variation include strain and meta-heterogeneity.•Conserved regions of hemagglutinin are similar in glycosylation distribution.

Variation of site-specific glycosylation measurements is not well understood.

Glycopeptide abundance distribution spectra can quantitatively describe variation.

Factors that produce the least variation include replicates and batches.

Factors that produce the most variation include strain and meta-heterogeneity.

Conserved regions of hemagglutinin are similar in glycosylation distribution.

Site-specific glycosylation analyses use measurements from high-resolution mass spectrometry and are often reported with no knowledge of their reproducibility or causes of variation. This study demonstrates how reproducible these measurements are and by what degree certain factors contribute to variation. Factors such as replicates and production batches result in very little variation, whereas viral strain and meta-heterogeneity contribute greatly to variation. Results are also reported here which compare conserved and nonconserved regions of influenza glycoproteins and different unrelated glycoproteins.