enGenes’ now offers three antigens that allow for differentiation of natural infection (N) vs. vaccine induced immunity (non-N, but RBD and/or Spike). The antigens are for use in different test settings and enGenes Biotech is offering its X-Press expression platform to enable rapid scale up to commercial bioreactor scale to make testing kits available across the EU.
Which protein to target?
Most commercially available antibody tests target the nucleocapsid protein (NP), as the most common virus protein of the pandemic pathogen SARS CoV-2. NP is used in diagnostic “antibody tests” used to detect various stages of infection SARS CoV-2 coronavirus infection, including COVID-19.
However, currently available vaccinations do not stimulate an immune response against the nucleocapsid protein, which is an indicator of developed immunity due to natural infection rather than vaccination.
Different proteins need to be targeted to set up appropriate serological tests to monitor the vaccination process and the development of non-N immunity in the population.
RBD and spike protein
One of these is the Receptor Binding Domain (RBD), the part of the spike surface protein that interacts with the ACE2 receptor, the entry point of SARS CoV-2 on the cell surface of human cells.
The other on is spike protein (S1 + S2 ectodomain of the SARS CoV-2 spike protein), located on the virus surface and mediating the virus’s uptake into human cells.
Since the currently available vaccines exclusively use the spike protein’s blueprint, a successful vaccination can be monitored using a corresponding antibody signal against RBD and/or spike protein.
Efficacy confirmed by studies
enGenes Biotech joined a consortium of other Vienna-based companies, with Technoclone and NovaSign, along with academic partners BOKU, ACIB, AIT, Vetmed, and MedUni Vienna, to investigate characterization and production of antigens for COVID-19 antibody assays.
The results of the study ‘A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays’, published on EBioMedicine.com, describes two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and NP. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers.
The study´s results clearly show that antigen quality has a significant influence on test performance. The ELISA test developed within this study has a specificity of 99.8% and is commercialized as a ready-to-use test format by Technoclone as Technozym NP and RBD ELISA).
NP production method
enGenes scientists Dr. Juergen Mairhofer and Dr. Gerald Striedner have contributed to a research study to be published in Analytical Chemistry that details a new method for expressing the NP protein.
The study ‘Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli’ show how interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) method could be established for in-process control, in particular to monitor product fragmentation and multimerization throughout the purification process. High titers of the nucleocapsid protein were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of chromatographic steps. This optimized process succeeded in producing 730 mg purified NP per liter of fermentation, corresponding to yield of 77% and with purity of 95%, confirmed by peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay. The HIC-MALS method enables purity monitoring with simultaneous access to molecular mass.
Rapid scale up
enGenes Biotech is now seeking partners to develop SARS CoV-2 variant proteins or other innovative approaches to help to control the pandemic. It is offering its proprietary enGenes-X-press technology as a manufacturing process with verifiable yields that can be scaled up to large bioreactor production within a short timeframe.
This would allow commercial quantities to be reliably produced from Austria for any application, accelerating vaccination programs across the EU.
About enGenes Biotech
enGenes Biotech GmbH (enGenes) is a contract research, development and manufacturing company that provides leading edge technologies and production services focused on recombinant proteins in bacteria. The company’s mission is to provide cost-effective and scalable production of recombinant proteins at a fraction of current cost, allied to a vision of developing a world-class portfolio of cutting-edge protein production technologies, relevant to a broad spectrum of application fields.
enGenes has developed advanced technologies to drive more cost-effective recombinant protein production processes, including its proprietary enGenes-X-press™ E. coli platform that achieves outstanding yields of soluble and active recombinant protein. enGenes-X-press™ has been successfully applied for the manufacturing of enzymes and biopharmaceutical products that failed to give economically feasible yields in a conventional expression host.
enGenes Biotech offers development and manufacturing services tailored around the needs of pharmaceutical and industrial biotech companies. The services include expression strain and vector development, fermentation process development and optimization, downstream process development, production of purified protein, technology transfer and scale-up support with technology out-licensing and co-development opportunities.
Resources
Click on A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays to read full study.
Click on Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein in E.Coli to access study.
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