Unix SEC C 300 UHPLC Analytical Column for Analysis of Prefusion Protein Stabilization for Paramyxovirus Vaccine Design

Product summary used in the paper - Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein

Paper Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11143371/

Introduction:

Unix SEC columns are unique to the industry with its encapsulated surface coat chemistry, surface area, and pore distribution. The particle size is also 1.8μm allowing for the best possible efficiency by limiting mass transport distance thus increasing the diffusion kinetics. The C series surface coat encapsulates the entire particle allowing for greater compatibility with sticky or hydrophobic molecules. Some other surfaces like BEH may be too hydrophobic for this application.

Researchers have stabilized different proteins in effort to induce higher immune responses in vaccine candidates. The stabilization of such regions allows for longer half-life and optimal presentation to generate effective neutralizing antibodies. Antibodies generated toward these stabilized fusion proteins have greater efficacy vs. the wildtype vaccine candidate. Researchers mentioned that the virus would require prefusion (preF) modification to generate an effective vaccine. SEC used was used to assess molar mass, dispersity, and retention.

Unix C -300 4.6 x 150mm was used to detect the different engineered regions in the RV1/3 in paramyxovirus. ?The substitution of Ser residues (hydrophilic) located at positions 470 and 477 for Val residues (nomenclature +S470V +S477V) increased stability of trimer. The modification with serine increases the hydrophilicity which plays a role in increasing coil stability. This stabilization prevented the generation of unwanted GCN4 (activated transcription factor) antibodies, Antibodies generated against GCN4 would significantly weaken the response.

SEC Method:

HPLC Column – Unix-C-SEC-300 4.6 x 150mm – Sepax Technologies

System – UHPLC Vanquish, Thermofisher

Detector – μDAWN TREOS/Optilab μT-rEX RO, inline Nanostar DLS

Sample Load – 10μg mass or 20μL volume

Temp- 25°C

Mobile Phase – 150mM sodium phosphate, 50mM NaCl, pH 7.0

Flow Rate – 0.35ml/min

Calculation – Chromeleon 7.2.8.0 and MALS data using Astra 8.0.0.19 using a dn/dc value of 0.185 For protein and 0.1410 for glycan. MW using RI for concentration and mass recoveries via UV 260nm

Results:

?Figure 1: Stabilized trimer head comparison with SEC:

Graph C in Figure 1 shows trimer (T) stabilized head - which is the 3-serine substituted for valine variant, while the blue monomer (M) is the unstabilized or wild type (minimal vaccination response).?

Graph D shows the MALS trace of the stabilized head trimer vs. the serine modified. The chromatograms reveal the molar mass, hydrodynamic radius, and dispersity of the trimer. The molar mass of the head stabilized trimer is slightly larger than the serine modified counterpart. The main reason for the difference may have to do with the conformation of the trimer and its effect on the hydrodynamic radius (the black trace with only the stabilized head). SEC RT shifts are more sensitive the to molecules net shape and the space it can occupy with minimal steric hindrance.

Researchers further investigated the effects of aa modification on stability and looked at each construct using SEC:

?Figure 2 – AA Modified Trimer Stability

The graphs below look at the effect of temperature each construct proving +S470V+S477V is the most stable variant/antigen.

RV 3 stabilization with modifications (S41P, Q89M, Q222I, N167P, L168P, F335P, S470V and S477V) for pre-fusion protein were also studied. SEC-MALS with Unix C revealed that trimerization wasn’t present in the wildtype vs. the modified protein (only Ecto 2P). Molar mass determined empirically at 156Kda was close to the expected mass of 156.3 proving that the SEC-MALS method is accurate and reliable.

Figure 3 – Wildtype vs Ecto2P Modified preF (perfusion f) protein.

The technique for stabilization applied to the RV 3 (perfusion f protein via the random coil )? was used to stabilize other paramyxoviruses. The researchers mention that the region for transformation in this virus was highly conserved and thus be successfully for other candidates or serotypes.

Researchers looked at another variant with substitutions (A44P, E170P, Q171P, S473V and A480V) ?in the hairpin loop region. They found an increase in antibody binding with this modification. The chromatograms below in Figure 4 show that the calculated value of 142 was within 2% of the expected value.

Figure 4 – Stabilized Trimer (T) vs. MALS

Conclusions:

The results from the paper using the Sepax UNIX C 300 UHPLC column demonstrate that the SEC-MALS method can accurately detect protein sequence modification in preF protein. Stabilization of this protein (coil and hair pin beta region) is critical for future vaccine development. This region of viral transformation is highly conserved and thus modifications within this domain can prove valuable for other serotypes. The researchers of this paper discovered the key region responsible for this transformation and techniques to stabilize it with amino acid substitutions in precise locations altering the conformation with changes in hydrophobicity. These changes increase the expression of neutralizing antibodies which are critical to preventing RSV induced virulence.

Article Summary: SEC-MALS with UNIX C 300 allowed for:

1) Low Shed

2) Surface Hydrophilicity

3) Measurement of preF aa engineered mutants

4) Assessed the stability of engineered constructs via temperature variation.

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Sincerely,

Anthony Sokol, 717-615-6354