Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry

Abstract

Modern analytical approaches employing high-resolution mass spectrometry (MS) facilitate the generation of a vast amount of structural data of highly complex glycoproteins. Nevertheless, systematic interpretation of this data at different structural levels remains an analytical challenge. The glycoprotein utilized as a model system in this study, human chorionic gonadotropin (hCG), exists as a heterodimer composed of two heavily glycosylated subunits. In order to unravel the multitude of glycoforms of recombinant hCG (drug product Ovitrelle), we combine established techniques, such as released glycan and glycopeptide analysis, with novel approaches employing high-performance liquid chromatography-mass spectrometry (HPLC-MS) to characterize protein subunits and native MS to analyze the noncovalent hCG complex. Starting from the deconvoluted mass spectrum of dimeric hCG comprising about 50 signals, it was possible to explore the chemical space of hCG glycoforms and elucidate the complexity that hides behind just 50 signals. Systematic, stepwise integration of data obtained at the levels of released glycans, glycopeptides, and subunits using a computational annotation tool allowed us to reveal 1031 underlying glycoforms. Additionally, critical quality attributes such as sialylation and core fucosylation were compared for two batches of Ovitrelle to assess the potential product variability.

Figure 1

Mirror plot of deconvoluted mass spectra of dimeric hCG of Ovitrelle batches BA059433 and BA056714. Spectra were obtained with an instrument resolution setting of R = 17 500 at m/z 200. Colored arrows represent the peak height of the highest peak in the signal series of the mirrored batch.

Figure 2

Annotation of hCGα glycoforms. Deconvoluted mass spectra of hCGα obtained by HPLC-MS analysis (a) after desialylation or (b) without enzymatic treatment. Glycoforms are indicated by letters (A–D). Red asterisks indicate fucose variants. Numbers above the peaks refer to Neu5Ac residues. Peak lists with all possible glycoform assignments for untreated and desialylated hCGα are available in Supporting Information, Annotations hCGα BA059433. The instrument resolution setting was 140 000 at m/z 200.

Figure 3

Annotation of hCGβ glycoforms. Deconvoluted mass spectra of carbamidomethylated hCGβ obtained by HPLC-MS analysis (a) after sialidase and PNGase F treatment, (b) after PNGase F treatment, (c) after sialidase treatment, and (d) in untreated form. Glycoforms are indicated by letters (A–F). Red asterisks indicate fucose variants. The number of Neu5Ac residues is indicated by the numbers above the peaks. Lightning bolts indicate two peaks that are further discussed in the main text. Peak lists with all possible glycoform assignments for untreated and desialylated hCGβ are available in the Supporting Information. Annotations: hCGβ BA059433. The instrument resolution setting was 140 000 at m/z 200.

Figure 4

Annotation of dimeric hCG glycoforms. (a) Raw mass spectrum of dimeric hCG obtained by native MS. Charge states are indicated. (b) Deconvoluted mass spectrum of dimeric hCG. The glycoforms contributing most to peak abundance are indicated by colored symbols and represent a combination of glycans present on hCGα (A) and hCGβ (B–G). Numbers above these symbols correspond to the number of sialic acids present. The spectrum was obtained with an instrument resolution setting of R = 17 500 at m/z 200.