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Chromatography in the Vaccine Pipeline

Vaccine vials in a laboratory.
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As vaccine technologies evolve, sophisticated analytical tools are needed to support vaccine discovery, development and manufacturing. Consequently, a variety of techniques ranging from cell-based to biochemical assays and instrumental analysis are deployed to ensure product quality.1, 2 Ideally, the chosen analytical approach is expected to be sensitive, robust and reliable, enabling the identification, characterization and quantification of a variety of analytes, including impurities, present in the sample. One such technique is chromatography.

 

Chromatography in vaccine production


Liquid chromatography (LC) is a work horse in the analysis of therapeutic and immunogenic agents, such as vaccines, both during development and production. A number of methods have been reported to isolate, characterize and quantify the desired target molecules. There are however limitations with existing chromatographic methods that scientists are working to overcome through innovations in column chemistries, chromatographic techniques or use of detectors other than photodiode arrays, to analyze complex molecules in challenging matrices.

 

When asked about the innovations that have helped the most in analysis by LC, Dr. Caryn L. Heldt, director of the Health Research Institute, the James and Lorna Mack Chair in Bioengineering, Professor in the Department of Chemical Engineering, at Michigan Tech responded, “There are many aspects that affect speed, sensitivity and selectivity. Speed and sensitivity are being improved with new resin beads, as well as monoliths and membranes that reduce or eliminate diffusion. These increase speed and make the peaks narrower, thus improving sensitivity. Selectivity is being improved with new ligands that are selective. In addition, new methods to detect peaks are helping to improve sensitivity. New detectors using light scattering can aid greatly in detecting larger biomolecules. Using mass spectrometry (MS) has also improved detection and improves the speed of identifying peaks.”

 

LC in combination with a variety of detectors is used during the stages of vaccine development and manufacturing. Let’s consider some examples of chromatographic techniques that are being applied to the vaccine pipeline.

 

Characterization and quantification

Chromatographic techniques are used to characterize and quantify vaccines during discovery, development and quality control. Along with transmission electron microscopy (TEM) and dynamic light scattering (DLS), size-exclusion chromatography (SEC) has been used to characterize virus-like particles (VLP) developed to combat SARS-CoV-2.3 D-antigen, present in the Sabin inactivated polio vaccine (sIPV) formulation, has been quantified by SEC too.4 LC with tandem MS has been used to quantify capsid protein (L1) in multivalent HPV vaccines.5

 

Process monitoring

The ease of coupling LC to different detectors makes it a useful tool for monitoring the production process. In order to track process intermediates and the multivalent vaccine product during production, SEC with a series of in-line detectors, such as ultraviolet (UV), multi-angle light scattering (MALS) and refractive index (RI), has been used to measure the molecular weights, protein and polysaccharide concentrations.6

 

Purification

During the manufacturing process, purification of the target molecule is crucial. Chromatographic techniques such as affinity chromatography, fast protein liquid (FPLC), ion-exchange (IEX), reversed-phase (RP) or hydrophobic interaction (HIC) chromatography are used to isolate the pure product.7, 8 Weak anion exchange chromatography has been used to remove rHBsAg aggregates, a recombinant form of anti-hepatitis B virus surface antigen, during the production of a hepatitis B vaccine.9 Orthogonal techniques are coupled in a two-dimensional format to achieve the desired separation. RP LC has also been coupled with hydrophilic interaction chromatography (HILIC) to purify QS-21, a highly potent vaccine adjuvant, from a commercially available extract of Quillaja saponaria bark.10

 

Impurity analysis

Vaccine safety is ensured by monitoring for impurities using LC. Simple RP-high-performance liquid chromatography (HPLC) has been used for the quantification of residual valproic acid (used for cell transfection during manufacture) impurities in influenza vaccines.11 Triton X-100 and formaldehyde, used for the purification of viral vaccines, have also been quantified by a simple RP-HPLC-UV method.12

 

Stability testing

Real-time and accelerated stability of a hepatitis E vaccine have been studied using liquid chromatography-mass spectrometry (LC-MS) and other analytical techniques.13

 

Clinical studies

An HPLC-tandem MS method has been validated to identify tumor-associated antigens to support clinical studies.14

 

Challenges and opportunities


Although chromatographic techniques such as affinity chromatography and IEX have proven to be effective for the purification of biomolecules, they have some limitations. “The biggest challenge for the chromatography of many vaccines, unless they are protein subunit vaccines, are the size of the molecule,” says Dr. Heldt. “Large biomolecules are harder to characterize because they are complex. They also do not diffuse into chromatography beads, thus greatly reducing the surface area available for binding. The lab of Giorgio Carta showed this well in his 2015 paper.15 The VLPs were unable to penetrate the beads after 12 hours, whereas IgG saturated most of the beads in about 5 minutes.”

 

“For this reason, monoliths and membrane chromatography are becoming more popular for large biomolecules.”

 

For instance, the implementation of affinity chromatography on a commercial scale has been limited and expensive. However, development of new media and ligands for purification of vaccines is expected to improve industrial production by eliminating many of the downstream processing steps.16

 

Considering the benefits of using automation for reducing the turnaround time, Dr. Heldt explained, “One of the keys to improving cycle time is the reduction in analysis time. Online and inline analytics will greatly reduce cycle time. Currently, there are few, if any, online or inline analytics for viral vaccines. More are coming, especially in the area of Raman spectroscopy. Analytics are getting closer to identifying the product, product purity and product concentration with Raman.”

 

In silico models and artificial intelligence (AI) are being used to accelerate chromatographic purification of vaccines.17 The advantages and disadvantages of using LC coupled to a mass spectrometer to characterize and quantify antigens, such as inactivated viruses, VLPs, recombinant proteins and protein-polysaccharides, both in the presence or absence of adjuvants, have been reported.18 A quality by design (QbD) approach has been adopted to develop a rapid, sensitive and selective ultra-high-performance liquid chromatography (UHPLC) method for the quality control of 4CMenB vaccine.19

 

Conclusion


Rapid development and manufacture of large volumes of vaccines has been effectively supported by chromatographic techniques such as affinity chromatography, IEX and RPLC during the production pipeline. Chromatography is used to monitor process intermediates, purify the product during manufacture, characterize and quantify the product post-production, analyze for impurities and study the stability of the product.

 

Despite the number of different techniques and methods available for the analysis of vaccines, there is still room for improvements. “There are many unmet needs in vaccine analysis. We currently assess for total protein, specific proteins, size and DNA/RNA content. However, none of these tests alone prove you have the product you want. We do not have any clear methods to assess the quality and functionality of many of these large biomolecules,” Dr Heldt concluded.

 

Thus, the applications of chromatography in the vaccine pipeline to support the production of efficacious and high-quality products will continue to grow.

 

 

References


1.    Thompson CM, Petiot E, Lennaertz A. et al. Analytical technologies for influenza virus-like particle candidate vaccines: challenges and emerging approaches. Virol J. 2013;10:141. doi:10.1186/1743-422X-10-141  

2.    Bazhenova A, Gao F, Bolgiano B et al. Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis. Biophys Rev.2021;13221–246. doi:10.1007/s12551-021-00791-z 

3.    Arora K, Rastogi R, Arora NM, et al. Multi-antigenic virus-like particle of SARS CoV-2 produced in Saccharomyces cerevisiae as a vaccine candidate. bioRxiv.2020. doi:10.1101/2020.05.18.099234

4.    Shin WJ, Hara D, Gbormittah F, Chang H, Chang BS, Jung JU. Development of thermostable lyophilized Sabin inactivated poliovirus vaccine. mBio. 9:e02287-18. 2018. doi:10.1128/mBio.02287-18 

5.    Ning T, Sun S, Nie J, et al. Simultaneous quantification of major capsid protein of human papillomavirus 16 and human papillomavirus 18 in multivalent human papillomavirus vaccines by liquid chromatography-tandem mass spectrometry.  J Chromat A.2020;1619:460962. ISSN 0021-9673. doi:10.1016/j.chroma.2020.460962.

6.    Deng JZ, Lancaster C, Winters MA, Phillips KM, Zhuang P, Ha S. Multi-attribute characterization of pneumococcal conjugate vaccine by size-exclusion chromatography coupled with UV-MALS-RI detections. Vaccine.2022;40(10):1464-1471.ISSN 0264-410X, doi:10.1016/j.vaccine.2022.01.042

7.    Abdulrahman A, Ghanem A. Recent advances in chromatographic purification of plasmid DNA for gene therapy and DNA vaccines: A review. Anal Chim Acta. 2018;1025:41-57. ISSN 0003-2670. doi:10.1016/j.aca.2018.04.001

8.    Safavi A, Kefayat A, Sotoodehnejadnematalahi F, Salehi M, Modarressi MH. Production, purification, and in vivo evaluation of a novel multiepitope peptide vaccine consisted of immunodominant epitopes of SYCP1 and ACRBP antigens as a prophylactic melanoma vaccine. Int Immunopharma. 2019;76: 105872.ISSN 1567-5769. doi:10.1016/j.intimp.2019.105872

9.    Kimia Z, Hosseini SN, Siamak S, et al. A novel application of ion exchange chromatography in recombinant hepatitis B vaccine downstream processing: Improving recombinant HBsAg homogeneity by removing associated aggregates. J Chromat. B, 2019; 1113:20-29. ISSN 1570-0232. doi:10.1016/j.jchromb.2019.03.009  

10.  Yizhi Qi, Christopher B. Fox. A two-step orthogonal chromatographic process for purifying the molecular adjuvant QS-21 with high purity and yield. J Chromat A. 2021;1635:461705. ISSN 0021-9673.doi:10.1016/j.chroma.2020.461705

11.  Yang G, Wang X, Yang Y, Yang R, Daniel B. Gowetski, Q. Paula Lei. Quantitation of residual valproic acid in flu vaccine drug substance. J Chromat B.2020;1152:122235, ISSN 1570-0232, doi:10.1016/j.jchromb.2020.122235 

12.  Rajendar B, Reddy MVNJ, Suresh CNV, Gambheerrao SK, Matur RV. A reversed phase HPLC-UV method for the simultaneous determination of residual formaldehyde and Triton X-100 in vaccine products. J Chromat B. 2021;1184:122977. ISSN 1570-0232. doi:10.1016/j.jchromb.2021.122977

13.  Yin X, Wang X, Zhang Z, et al. Demonstration of real-time and accelerated stability of hepatitis E vaccine with a combination of different physicochemical and immunochemical methods. J Pharm and Biomed Anal. 2020;177: 112880. ISSN 0731-7085.doi:10.1016/j.jpba.2019.112880

14.  Ghosh M, Gauger M, Marcu A, et al. Guidance document: Validation of a high- performance liquid chromatography-tandem mass spectrometry immunopeptidomics assay for the identification of HLA Class I ligands suitable for pharmaceutical therapies. Mole & Cellular Proteom. 2020;19(3):432-443.doi:10.1074/mcp.C119.001652

15.  Wu Y, Abraham D, Carta G. Particle size effects on protein and virus-like particle adsorption on perfusion chromatography media, J of Chromat A, 2015;1375:92-100, ISSN 0021-9673, doi:10.1016/j.chroma.2014.11.083

16.  Zhao M, Vandersluis M, Stout J, Haupts U, Sanders M, Jacquemart R. Affinity chromatography for vaccines manufacturing: Finally ready for prime time? Vaccine. 2019;37(36):5491-550., ISSN 0264-410X. doi:10.1016/j.vaccine.2018.02.090    

17.  Keulen D, Geldhof G, Le Bussy O, Pabst M, Ottens M. Recent advances to accelerate purification process development: a review with a focus on vaccines. J Chromat A. 2022;463195.ISSN 0021-9673. doi:10.1016/j.chroma.2022.463195

18.  Hickey JM, Sahni N, Toth RT, et al. Challenges and opportunities of using liquid chromatography and mass spectrometry methods to develop complex vaccine antigens as pharmaceutical dosage form. J Chromat B.2016;1032:23-38. ISSN 1570-0232. doi:10.1016/j.jchromb.2016.04.001

19.  Nompari L, Orlandini S, Benedetta Pasquini B, et al. Quality by design approach in the development of an ultra-high-performance liquid chromatography method for Bexsero meningococcal group B vaccine. Talanta. 2018;178: 552-562.ISSN 0039-9140. doi:10.1016/j.talanta.2017.09.077

Meet the Author
Srividya Kailasam, PhD
Srividya Kailasam, PhD
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