Select language: Hrvatski English

Production process optimization of viral vaccines and gene vectors

Virus particles are used for medical applications as vaccines or viral gene therapy vectors. Viral vaccines are mainly based on attenuated or killed viruses (e.g. attenuated viruses; measles, mumps, rubella, poliomyelitis and killed viruses; poliomyelitis, influenza). Vaccines can also be based on purified virus proteins (e.g. vaccine against hepatitis B) or multiprotein complexes without genetic material called virus-like particles (e.g. vaccine against human papilloma virus). All these vaccines induce immune protection and do not cause disease. Gene therapy includes transfer of genetic material to cells to achieve therapeutic effect. Therefore, required genes are largely delivered by viral vector systems based on adenovirus, retrovirus (e.g. lentivirus), adeno-associated virus, herpes simplex virus, and Vaccinia virus.

Virus particles for vaccines and gene vectors are produced in cell substrates (in vitro) or in chick embryos (in vivo). This part of the production process – virus growth and harvesting is called the upstream processing. Harvested virus requires purification from contaminants originating from the growth medium (e.g. fetal serum albumin) or parts of host cells (e.g. host cell proteins and DNA) used for virus growth. Possible contamination are also particle-related impurities like free viral proteins, empty capsids and virus aggregates. Purification of virus particles after harvesting is called downstream processing. The aim of downstream processing is a final product of high efficiency and safety that complies with the strict regulations of health agencies.

Defining optimal production conditions for propagation of viruses with high yields and methodology for virus purification preserving virus infectivity and obtaining high recoveries is a special field.

Downstream virus processing accounts up to 70 % of the overall production costs and is a bottleneck of viral vaccines and gene vector production. Downstream processing includes several purification steps including centrifugation (a process of particle separation based on size, shape, volume and density under applied centrifugal force), various filtration processes (membranes of various pore size can be used to this aim, enabling passing of molecules smaller than pores and retention of larger ones), and chromatographic procedures (separation of crude virus suspensions, applied under pressure, on chromatographic matrices of various functionalities due to differential adsorption to matrix according to virus properties such as size, charge, hydrophobicity etc.) which are gaining more attention with the development of new chromatographic media. Based on the property used for chromatographic separation of viruses the mostly used modes of chromatography are: ion exchange, hydrophobic interaction, affinity and size-exclusion chromatography.

Viruses are very delicate macromolecular complexes and their infectivity can easily be lost, therefore downstream processing has to be optimized in order to maximize recoveries. Since biochemical and biophysical properties are specific for each virus and they determine the purification process, purification process for each virus requires optimization. In addition, thorough understanding of the entire purification process is crucial in order to define critical points that can influence the quality of the final product. In order to gain knowledge about the process Design of Experiments methodology is used that provides information about the influence of process parameters and their possible interactions on the process itself. Design of Experiments is a part of Quality by Design initiative that ensures product quality.

References:
Roldao et al. Virus-like particles in vaccine development. Expert Rev. Vaccines 9 (2010) 1149-1176.
Wolff & Reichl. Downstream processing of cell culture derived virus particles. Expert Rev. Vaccines 10 (2011) 1451-1475.
Segura et al. Downstream processing of oncoretroviral and lentiviral gene therapy vectors. Biotechnol. Adv. 24 (2006) 321– 337.
Nestola et al. Improved Virus Purification Processes for Vaccines and Gene Therapy. Biotechnol. Bioeng. 112 (2015) 843-857.