African Swine Fever (ASF) is an acute, hemorrhagic and highly contagious disease caused by African Swine Fever Virus (ASFV), which infects domestic pigs and wild boars. The mortality rate of peracute and acute ASF infections can reach 100%. Its clinical signs resemble those of classical swine fever, and laboratory testing is the only definitive diagnostic method. The World Organisation for Animal Health (WOAH) lists ASF as a notifiable terrestrial animal disease, and it is also classified as a Class I major animal epidemic requiring strict prevention and control in China.
As the sole member of the Asfarviridae family, ASFV is a large double-stranded DNA virus with a genome of approximately 175–190 kb. It encodes numerous structural proteins as well as proteins that facilitate viral evasion of host immune defenses. The virion adopts an icosahedral shape with a five-layer structure and a diameter of around 200 nm. Its sophisticated viral structure and complex pathogenic mechanisms have greatly hindered the research and development of ASF vaccines.
Globally, major R&D routes for ASF vaccines include inactivated vaccines, live attenuated vaccines, subunit vaccines and viral vector vaccines. Inactivated ASF vaccines were among the earliest candidates studied, but they fail to provide sufficient protective efficacy and have been largely phased out. Live attenuated vaccines carry potential safety and stability risks such as virulence reversion, immunosuppression and latent infection.
Subunit Vaccines
ASF subunit vaccines induce humoral and cellular immunity by delivering protective antigens (e.g., P30, P54, P72). Featuring high biosafety and the capability to differentiate vaccinated animals from infected ones, they have emerged as a promising strategy against ASF. Manufactured via recombinant DNA technology to express viral surface antigens, followed by purification and inactivation, these vaccines deliver favorable safety and efficacy profiles. The core production workflow covers antigen screening and gene cloning, protein expression and purification, inactivation, adjuvant formulation and quality control verification, ultimately yielding vaccine formulations capable of triggering specific immune responses.
Viral Vector Vaccines
Live viral vector vaccines use attenuated viral vectors to deliver viral antigens and elicit immune responses. Their advantages include robust immunogenicity and long-term protective immunity with a single dose. However, pre-existing immunity against the vector may compromise vaccine efficacy. Additionally, this technology is hampered by low production efficiency, high costs and potential risks of viral genome integration.
Harvest and Clarification
Cell culture harvests contain abundant target products along with impurities such as cell debris and metabolic by-products, resulting in turbid feedstocks. Direct concentration of unclarified harvests will rapidly foul ultrafiltration membranes, disrupting the concentration process and even causing permanent membrane damage. Therefore, clarification is an indispensable pre-concentration step to remove large particulate contaminants and obtain translucent or clear feedstocks, which ensures smooth concentration operations and extends the service life of ultrafiltration membranes. High-performance clarification processes are required to achieve high particulate removal efficiency and high product recovery, allow easy scale-up, and protect downstream processing equipment.
Centrifugation is commonly used to remove large particles, followed by multi-stage depth filtration (e.g., 1.2 μm → 0.45 μm) to clarify the supernatant. For large-scale production, centrifugation is time-consuming, while depth filtration consumes large quantities of filter media and drives up operational costs. In high-density cell culture processes, depth filters exhibit reduced loading capacity, further increasing costs and leading to excessive product dilution.
Tangential Flow Filtration (TFF) operates under transmembrane pressure: substances and impurities smaller than the membrane pores pass through, while large particles such as cells are retained, making it well-suited for large-scale manufacturing. TFF membrane modules are mainly available as flat-sheet cassettes and hollow fiber membranes. Microfiltration for clarification typically adopts membranes with pore sizes of 0.45 μm or 0.22 μm. Hollow fiber tangential flow microfiltration can directly process high-solids feedstocks such as high-density cell culture harvests, eliminating the need for centrifugation and pre-filtration. This streamlined process features simple operation, and the membranes can be cleaned and reused repeatedly, cutting down capital investment and operational costs while adapting to automated large-scale production.
Ultrafiltration Concentration and Buffer Exchange (TFF)
TFF-based ultrafiltration enables hundreds-fold concentration of protective antigens and effective removal of host cell proteins. Hollow fiber membranes stand out for ultrafiltration and purification due to their gentle low-shear operation, low fouling tendency, flexible application, long service life, cost-effectiveness and excellent scalability.
Selecting an appropriate membrane pore size is critical to concentration efficiency and final product quality. The chosen membrane must fully retain target molecules to guarantee high recovery, while delivering satisfactory removal of impure proteins and reasonable processing throughput. The core selection principle is to use the largest possible uniform-pore membrane that can retain the target molecules.
