Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay

Riches-Duit, Rebecca; Hassall, Laura; Kogelman, Amy; Westdijk, Janny; Rajagopal, Shalini; Davletov, Bazbek; Doran, Ciara; Dobly, Alexandre; Francotte, Antoine; Stickings, Paul

doi:10.1016/j.biologicals.2020.12.002

Cited by 9 publications

(5 citation statements)

References 29 publications

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“…Notably, mAb 3-14 was shown to have relatively poor sensitivity to heat degradation of DTxd, with binding signal only slightly reduced for heat treated compared to intact DTxd. Upon exposure to heat, unfolding of the toxoid can occur, and this has the potential to affect both the integrity of conformational epitopes as well as the accessibility of individual epitopes on the molecular surface [ 10 , 22 ]. The 60 °C heat treatment represents a thermally accelerated degradation condition that is known to cause a reduction in DTxd vaccine potency in animal protection models ([ 23 ] and confirmed in house (data not shown)).…”

Section: Resultsmentioning

confidence: 99%

“…BLI is a real time technology that uses the interference pattern of white light reflected from the surface of a biosensor where molecules are attached to assess kinetics of mAb-epitope binding [ 9 ]. Biosensor-based methods have been used to support a variety of vaccine research, development, and production studies including vaccine quality control [ 10 ], epitope design and epitope binning [ 11 ], characterization of pathogen mechanism of action and host immune response [ 12 ], antibody affinity [ 13 ] and therapeutics and clinical studies [ 14 ]. In this study, BLI was used for the final SEAT method, wherein immobilized mAbs on the biosensor are used to bind free, unlabeled DTxd in solution, as a means of assessing individual epitopes.…”

Section: Introductionmentioning

confidence: 99%

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Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes

et al. 2022

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In the vaccine industry, multiple physicochemical, immunological, in vitro and in vivo analytical methods are applied throughout the manufacturing process to characterize and monitor the quality of vaccines. Presented here is the Single Epitope Antigenicity Test (SEAT), an innovative, quantitative epitope profiling method which provides an extended immunochemical analysis for diphtheria toxoid (DTxd) to be used for consistency testing during manufacturing process changes. The method uses BioLayer Interferometry (BLI) and a panel of monoclonal antibodies (mAbs) to independently assess nine individual antigenic sites of DTxd. The panel includes mAbs which are functional, bind distinct sites on DTxd and are able to distinguish intact DTxd from that which has been exposed to heat treatment. The SEAT method was qualified for precision, accuracy, and linearity, and was used to define a preliminary comparability range for DTxd made using the current manufacturing process. DTxd lots manufactured using alternate processes were assessed in the context of this range to determine the impact on DTxd antigenicity. Epitope profiling by SEAT provides quantitative information on the integrity of multiple important antigenic regions of DTxd, and therefore represents a valuable tool in a comprehensive analytical test package which can be used to support manufacturing process changes for vaccines.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes

et al. 2022

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“…For diphtheria vaccines, antigen quantity and integrity as well as the degree of adsorption to adjuvant are critical quality attributes to be monitored. Within the VAC2VAC project we have developed a capture ELISA, utilizing mAbs that recognize functional epitopes on the DTxd (Riches-Duit et al, 2021), to measure Tab. 5: Comparison of curve shapes for different products against NIBSC standards and drug substances Any differences observed in the slope ratios (as indicated by a slope ratio < 0.9 or > 1.11) or in the asymptotes (as indicated by an asymptote difference of < -0.05 or > 0.05) are highlighted in grey.…”

Section: Discussionmentioning

confidence: 99%

“…The two anti-diphtheria mAbs selected for ELISA development were DT05 (a rat IgG2a antibody provided by the National Institute for Biological Standards and Control, NIBSC, UK) and Dim9 (a mouse IgG1 antibody provided by Intravacc, The Netherlands) (Riches-Duit et al, 2021). The mAbs were produced by hybridoma culture and purified by protein A/G affinity chromatography and were buffer exchanged into phosphate-buffered saline (PBS) for storage.…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines

Hassall¹

2024

ALTEX

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the production of toxin-neutralizing antibodies. They are among the most widely employed and successful human vaccines, and their use has significantly decreased the incidence of the disease worldwide (Clarke et al., 2019), although there can still be significant outbreaks where there is insufficient vaccination coverage.Diphtheria vaccines consist of formaldehyde-inactivated preparations of DTxn (forming diphtheria toxoid, DTxd) adsorbed onto an aluminium adjuvant (aluminium hydroxide, Al(OH) 3 , aluminium phosphate, AlPO 4 , or a combination of both), and include additional non-diphtheria components such as tetanus toxoid, pertussis (acellular antigens or whole cell), inactivated poliomyelitis, hepatitis B surface antigen, and Haemophilus influenzae type b polysaccharide. The chemical inactivation of DTxn results in toxoids that consist of 1 IntroductionDiphtheria is a potentially fatal infection caused by toxigenic strains of corynebacteria, primarily Corynebacterium diphtheriae. Diphtheria is generally an acute respiratory infection, characterized by the formation of a pseudomembrane in the throat, but cutaneous infections are also possible. Diphtheria toxin (DTxn), an exotoxin that inhibits protein synthesis and causes cell death, is the most important virulence factor for disease-causing C. diphtheriae strains. As well as causing local tissue destruction, DTxn can enter the bloodstream and disseminate throughout the body causing complications such as myocarditis and neuropathy (Sharma et al., 2019

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“…To avoid the issue of high variability in in vivo studies and to adhere to the “3R” principle (reduction, replacement and refinement of animals) [ 9 ], several in vitro potency assays have been developed as alternatives [ 10 ]. ELISA (Enzyme-linked immunosorbent assay)-based in vitro assay is one of them, and such assays have been evaluated both for licensed and developmental vaccines against multiple pathogens, such as human papillomavirus [ 11 ], hepatitis B [ 12 ], rabies [ 13 ], diphtheria [ 14 ], hepatitis E [ 15 ] and respiratory syncytial virus (RSV) [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

Development and Qualification of an Antigen Integrity Assay for a Plasmodium falciparum Malaria Transmission Blocking Vaccine Candidate, Pfs230

Miura

Pham²,

Lee³

et al. 2022

Vaccines

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During development of a subunit vaccine, monitoring integrity of the recombinant protein for process development and quality control is critical. Pfs230 is a leading malaria transmission blocking vaccine candidate and the first to reach a Phase 2 clinical trial. The Pfs230 protein is expressed on the surface of gametes, and plays an important role in male fertility. While the potency of Pfs230 protein can be determined by a standard membrane-feeding assay (SMFA) using antibodies from immunized subjects, the precision of a general in vivo potency study is known to be poor and is also time-consuming. Therefore, using a well-characterized Pfs230 recombinant protein and two human anti-Pfs230 monoclonal antibodies (mAbs), which have functional activity judged by SMFA, a sandwich ELISA-based in vitro potency assay, called the Antigen Integrity Assay (AIA), was developed. Multiple validation parameters of AIA were evaluated to qualify the assay following International Conference on Harmonization (ICH) Q2(R1) guidelines. The AIA is a high throughput assay and demonstrated excellent precision (3.2 and 5.4% coefficients of variance for intra- and inter-assay variability, respectively) and high sensitivity (>12% impurity in a sample can be detected). General methodologies and the approach to assay validation described herein are amenable to any subunit vaccine as long as more than two functional, non-competing mAbs are available. Thus, this study supports future subunit vaccine development.

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Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay

Cited by 9 publications

References 29 publications

Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes

Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes

Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines

Development and Qualification of an Antigen Integrity Assay for a Plasmodium falciparum Malaria Transmission Blocking Vaccine Candidate, Pfs230

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