key: cord-287853-cob7ur35
authors: Sharma, Vaneet Kumar; Sharma, Ity; Glick, James
title: The expanding role of mass spectrometry in the field of vaccine development
date: 2018-05-31
journal: Mass Spectrom Rev
DOI: 10.1002/mas.21571
sha: 
doc_id: 287853
cord_uid: cob7ur35

Biological mass spectrometry has evolved as a core analytical technology in the last decade mainly because of its unparalleled ability to perform qualitative as well as quantitative profiling of enormously complex biological samples with high mass accuracy, sensitivity, selectivity and specificity. Mass spectrometry‐based techniques are also routinely used to assess glycosylation and other post‐translational modifications, disulfide bond linkage, and scrambling as well as for the detection of host cell protein contaminants in the field of biopharmaceuticals. The role of mass spectrometry in vaccine development has been very limited but is now expanding as the landscape of global vaccine development is shifting towards the development of recombinant vaccines. In this review, the role of mass spectrometry in vaccine development is presented, some of the ongoing efforts to develop vaccines for diseases with global unmet medical need are discussed and the regulatory challenges of implementing mass spectrometry techniques in a quality control laboratory setting are highlighted.

As the target populations for the vaccines are healthy individuals, pregnant women, or infants, vaccine safety is of paramount importance. Appropriately, the vaccine landscape is changing from traditional vaccine approaches to cost-effective, highly scalable, and safe recombinant vaccines. 9, 10 Using recombinant DNA technology, antigens are expressed in yeast, Escherichia coli, baculovirus expression vector system (BEVS), or mammalian cell lines. [11] [12] [13] Recombinant antigens are engineered to mimic the first step of virus attachment to the cell surface which is mediated by specific glycoproteins. The expressed recombinant antigens undergo multiple purification cycles to produce highly purified vaccines. 14, 15 In order for recombinant vaccines to be acceptable to regulatory authorities, in-depth analytical characterization needs to be performed In this review, we focus on the expanding role of mass spectrometry in vaccine development, irrespective of the route of production. We also highlight the regulatory challenges and limitations of mass spectrometry-based techniques which constrain its further implementation as a quality control batch release assay in cGMP manufacturing.

The role of mass spectrometry ( Alternatively, an O-linked glycan can be formed by linking the oligosaccharide to a serine or threonine residue. Glycosylation play a fundamental role in antigen conformation, folding, stability, solubility, and importantly, immune response. 16, 17 Plants, yeasts, and non-human cell lines generate glycans that are not compatible and bioactive within human hosts. 18 Thus, about 70% of all recombinant glycoproteins are produced in mammalian-based expression systems such as Chinese Hamster Ovary (CHO) cells. 19 Analytical characterization of a glycoprotein is challenging as there is inherent unpredictability associated with the glycans; they are either macroheterogeneous (potential glycan site in the protein not glycosylated) or microheterogeneous (different glycan structures found on the same site in the expressed protein). Glycosylation analysis is performed to understand the nature of structural heterogeneity of glycans, quantify them, and more importantly to determine where glycosylation occurs (site specific analysis). Mass spectrometry based techniques are valuable tool for detecting and investigating glycosylation ( Figure 2 ). Although MS-based glycosylation analysis is not without limitations, the advances in MS instrumentation and glycan analysis software have led to increased resolution, automated identification, quantitative determination, and accurate structural characterization. 16, 20, 21 Another critical quality attribute (CQA) of glycoprotein is intramolecular disulfide bonds (S-S linkages); they ensure correct folding, functional activity and stability. Incorrect formation of disulfide bonds can cause protein misfolding which tends to promote aggregation which could result in an unwarranted immune response.

Confirmation of correct disulfide bond formation in the recombinant First study analyzing the site-specific Nglycosylation of gp120. This report describes the structural characterization of the expressed gp120.

Reversed phase HPLC of the tryptic digest. Peptides collected from RP-HPLC were further identified by amino acid analysis (AAA) or Nterminal sequencing analysis.

Leonard et al 38 HIV-1 gp120 Expressed in CHO Cells.

Mass spectrometric characterization of the glycosylation pattern. Of HIV-1 gp120.

MALDI and nanoESI-LC-MS/MS using a hybrid quadrupole-time-of-flight tandem mass spectrometer (Q-TOF) was used to assign glycosylation sites.

Zhu et al 111 HIV gp140 JR-FL and CON-S Env expressed in CHO and HEK293 cells.

A glycopeptide-based mass mapping approach was used to characterize the glycosylation of two Env protein vaccine candidates in a glycosylation site-specific fashion. Mass spectrometry based approach was used to map the complete glycosylation profile at every site in eleven HIV-1 Env trimers.

High-resolution LC-MS/MS was performed using an Orbitrap Velos Pro hybrid mass spectrometer (Thermo Scientific) equipped with ETD and coupled to an Acquity UPLC system (Waters).

Go et al 49 HIV gp120 Monomers of the BG505. SOSIP.

Global N-Glycan site occupancy of HIV-1 gp120 by metabolic engineering and high-resolution intact mass spectrometry was performed.

Released N-glycan analysis was carried out using Synapt G2Si ion mobility mass spectrometer. Native highresolution mass spectrometry was performed on Q Exactive hybrid Quadrupole-Orbitrap mass spectrometer. As illustrated in the following section and in Table 1 , mass spectrometry-based techniques have been used to perform the structural characterization, glycosylation profiling and antigen quantitation during the development of the HIV, influenza, Dengue, Ebola, Meningococcal, and other vaccines. The review also highlights that mass spectrometry-based methods such as glycan analysis has been used to analyze a specific envelope glycoproteins (Env) and has broad applicability to any other glycoprotein-based vaccines.

The quest for a safe and effective vaccine to protect against human One of the hypotheses being explored for protective immunity against HIV is to induce broadly neutralizing antibodies (bNAbs) that target the highly glycosylated HIV-1 virion-associated Envelop. 33 A number of approaches are being pursued in the HIV-1 vaccine development field to establish protective immunity, including monomeric gp120 subunits and oligomeric gp140 and trimeric envelope glycoprotein to elicit bNAbs. [34] [35] [36] Each of these Envelop (Env)

immunogens have multiple exposed epitopes and are heterogeneously glycosylated molecules, with more than 50% of their mass consisting of glycans (∼28 N-linked glycans). It is critically important to monitor this HIV Env glycan shield during vaccine development. 37 Crispin and collaborators also extensively investigated the native glycosylation profile of Envs, in particularly, trimeric immunogen BG505 SOSIP.664. [50] [51] [52] [53] Their studies led to an increased understanding of the glycan shield of the Env and it is now largely accepted that to induce bNAbs, the HIV vaccine candidate should have a glycan profile similar to the one present on the native Env trimers. [54] [55] [56] In a thorough study on trimeric Env glycosylation, Behrens et al 51 

Influenza virus is a segmented, enveloped RNA virus and is among the IC-IDMS-MRM method is truly an alternative to the approved SRID method as it has dual purpose "potency and content determination", was found to be equivalent to the SRID method and can also be used in response to a pandemic influenza threat. 75, 76 Additionally, a direct Ultra-Performance Liquid Chromatography (UPLC)-IDMS method was reported for the rapid and accurate quantification of influenza NA. 77 Label-free MS-based methods have also been reported for the simultaneous identification and quantification of HA and NA in influenza vaccine with samples analyzed by LC-MS E on a Waters Synapt G2 mass spectrometer. 78 Quantification of proteins by labelfree LC-MS E is a powerful tool, in this method, alternating scans of low collision energy and elevated collision energy during LC-MS analysis to obtain both protein identity and quantity in a single experiment.

Quantification based on the experimental data showed that the signal intensity was proportional to concentration which allowed for the amount of any protein in the mixture to be estimated. LC-MS E utilizes parallel, multiplex fragmentation where all peptide precursors are simultaneously fragmented throughout the chromatographic The SRID assay has been used for over 40 years as a quantitative and potency method throughout the world despite issues regarding variability and availability of standard reagents. Thus, even though new methods like HPLC and mass spectrometry are being developed, it will take some time for these methods to be adopted worldwide. As the next influenza pandemic cannot be predicted, the health authorities' pandemic preparedness efforts include efforts to ensure expedited availability of pandemic vaccines. Methods such as HPLC and MS, with their ability to quantitate antigens without standard reference reagents,

can become a cornerstone of pandemic influenza preparedness.

The viral genus Flavivirus, includes Dengue virus, yellow fever virus, and and 153 were glycosylated and, predominately, the N-glycan at Asn67 was a high mannose-type and at Asn153 was mainly a combination of complex-and hybrid-type glycans. 87 This study provided important new insights for the role of glycans in the dengue virus-host cell interactions.

In a separate study, accurate quantitation of the expressed four viral particles in the tetravalent dengue vaccine (CYD) was performed using targeted MS in selected reaction monitoring (SRM) mode. 88 The study described an orthogonal quantitation strategy (targeted MS in SRM mode) and demonstrated that the variability of the MS method was low (between 8% and 17%) and the assay was linear between 6.25 and 200 nmol/L. Based on the reported method performance, it could be used to release future batches of the tetravalent dengue vaccine.

Ebola The data presented demonstrated that the N-glycan patterns were similar between GP 1,2 but O-glycan patterns were remarkably different on GP 1,2 the five Ebola viruses. This study should serve as the foundation for future Ebola viral entry and immunogenicity studies. 91 To improve on the conventional approaches for absolute quantitation of GP1 in Ebola virus-like particles (eVLPs), an isotope dilution full-scan liquid chromatography-high-resolution mass spectrometry method was developed using an UltiMate 3000 HPLC and an

Orbitrap Elite Hybrid Ion Trap-Orbitrap mass spectrometer. 92 The reported MS quantitation method provided not only a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1 but also ensured adequate preclinical/clinical dosing.

Chikungunya is a mosquito-borne viral disease, endemic in Africa and

Southeast Asia and has also recently emerged in the Caribbean.

Currently there are no drugs or vaccines available for treatment or prevention. The name "Chikungunya" derives from a Makonde word meaning "to become contorted," and describes the stooped appearance of sufferers with joint pain (arthralgia MS-based approaches have been used to perform the physicochemical characterization for all of these vaccines. 96 In one of the studies, researchers used LC-MS to characterize glycosylated lysine residues in

Menveo®. 97 In another study a LC-MS method was used to determine the relative reactivity of lysine residues in CRM 197 to determine which of these amino acids were more susceptible to conjugation. 98 LC-MS was also used to quantify the Bexsero® vaccine which was the first vaccine developed by reverse vaccinology; a genome-based approach to vaccine development. 99 in the field of vaccines, Vera-Velasco et al not only quantified the viral F, G and M proteins present in the viral particles but also analyzed the cellular proteomic composition in the NiV vaccine candidate.

Traditionally, viral particles have been described as pure entities carrying only viral-derived proteins but the authors successfully analyzed the ratio between cellular and viral proteins in the NiV vaccine candidate using LC-MS/MS. 108

West Nile fever is a flavivirus that causes a viral infection typically spread by mosquitoes. To date, no vaccine is available to prevent the West Nile infection. Partially purified virus-like particles were resolved by SDS-PAGE, and the Coomassie blue-stained band corresponding to Env protein was excised from the gel, destained, and analyzed by MS.

Microcapillary LC-MS was performed using a LCQ DECA ion-trap mass spectrometer (Thermo Finnigan) to identify the presence of Env protein in virus-like particles to help in developing a potential vaccine. 109

In should be a supplemental batch release test to ensure that the product is within specifications and without any unwarranted modifications.

Using MS techniques will ensure improved quality and increased safety for clinical trial participants.

In conclusion, the evidence suggests that the emergence of MS-based techniques has advanced the field of vaccine development. 

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