RSV vaccine purification schemes

Jan 08,2025

Bestchrom

Human Respiratory syncytial virus (RSV) is A non-segmental single-strand negative strand RNA virus. The virus belongs to paramyxoviridae, pneumovirus genus and pneumovirus subfamily. It is divided into two subtypes, specially type A and type B. The virus is seasonally prevalent: A and B subtype will alternate or co-occur.

RSV has a total genome length of 15.2Kb and encodes 11 viral proteins, among which three proteins are present on the viral envelope. Specifically, adhesion protein G, fusion protein F and small hydrophobic protein SH. G and F proteins play a vital role in the process of viral infection. Thus, G and F proteins are mainly targeted for antigen design in RSV vaccine development.

Currently, RSV vaccine R&D approaches can fall into the following types: recombinant live attenuated virus (LAVs) vaccine, recombinant subunit vaccine, mRNA vaccine and adenovirus vector vaccine.

RSV recombinant live attenuated virus

In the absence of adjuvant , recombinant live attenuated vaccines (LAVs) induce an immune response similar to that of natural infection by the pathogen, thus inducing a higher immune response compared with other types of vaccines. Such LAVs can be found in vaccines from many brands including BLB-201 of Blue Lake Biotechnology, CodaVax-RSV of Codagenix and VAD00001 of Sanofi Pasteur.

The production of vaccine requires the capture of live attenuated virus, which can be achieved by the following process:

Process captured virus solution with nuclease and filtration & clarification, the solution can be purified with Diamond Layer 700 resin to remove the host cell protein (HCP) and nucleic acid. The process enjoys advantages including high sample loading volume (can reach 10CV) and high yield(50%-60%), ensuring the purity and activity of virus.

Fig.1 RSV recombinant live attenuated virus purification process

RSV recombinant subunit vaccine

R&D institution will usually choose G or F proteins in the surface of RSV to design antigen molecules. The absence of complete virus genome brings the safety of vaccine to a new level, making it the ideal choice for people with compromised immune function including senior citizens. G protein based vaccine includes RSV virus from Advaccine, F protein based vaccine includes Arexvy from GSK and Abrysvo from Pfizer's bivalent RSV vaccine.

Take the F protein preparation for example, the following process can be used:

After CHO cell culture, harvest supernatant and clarify. The first stage uses Diamond Virus-S resin to capture and purify RSV Pre F protein while letting HCP flow through.

The second stage uses Diamond Q Mustang resin, which further removes impurities including HCP and HCD via binding-elution mode.

The third stage uses mixed-mode resin Diamond MIX-A, which removes product-related impurities and trace HCP via binding mode.

Then nano-filtration, ultrafiltration and bactericidal filtration were performed to obtain antigenic stock solution

Fig.2 the obtaining process of antigenic stock solution

RSV mRNA vaccine

mRNA, as vaccine platform, only carries genetic information from specific proteins. Due to the low possibility of interferon and modification on host genetic information, the vaccine enjoys high security level. mRNA-1325 vaccine from Moderna is a typical vaccine in this type. The production process of mRNA vaccine includes steps including plasmid template preparation, mRNA synthesis & purification as well as LNP preparation and sterile filling.

The preparation of plasmid can adopt the following three-step purification process.

Fig.3 Three-step purification process for plasmid preparation

Linearized plasmid DNA can be obtained by restriction enzymes. It is also possible to use Q Bestarose HP resin to remove relatively big host genomic DNA , plasmid DNA and some enzymes.

In the purification of mRNA RSV vaccine after in vitro transcription, mixed-mode resin Diamond Layer 700 can be used for initial capture stage, which will remove process-related impurities including pDNA, enzyme and NTP via flow-through mode.

Polishing stage can use strong CEX resin Diamond Q Mustang or BestPoly 30Q. Alternatively, HIC resin with high resolution Diamond Phenyl Mustang or Diamond Butyl Mustang can also be used for the removal of product-related impurities including segments or dsRNA.

Fig.4 Purification process flow of mRNA RSV vaccine

RSV vector vaccine

Major viral vectors used in RSV vaccine are adenovirus vector and poxvirus vector. The approved adenovirus vector COVID-19 vaccine has a relatively mature production process. Typical providers of RSV vector vaccine include Janssen and Bavarian Nordic.

Take adenovirus vector vaccine as example, the following process can be used for preparation and purification:

HEK293 cell culture and viral infection, cell lysis by surface activator, degradation of host cell nucleic acids by nuclease, lysate then is clarified by a deep filter, concentrated by 300kDa hollow fibers and change solution. The solution then will go through initial purification by Diamond Q Mustang and polishing by Diamond Layer 700. The feedstock then goes through 300kDa ultrafiltration, concentration and solution change, finally obtain the vaccine stock solution.

Fig.5 Process for Vaccine stock solution preparation

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