For reasons of patient safety and tolerance, host cell DNA in a final product must be reduced to appropriate levels. Other process elements such as aggregation and an excess of impurities can compromise viral particle recovery.įinal Product Purity Levels: Regulatory agencies provide recommendations and requirements regarding acceptable residual amounts of contaminants in final drug products. Thus, companies should take such factors into consideration when selecting depth filters because some depth filter devices include a 0.1-μm membrane that could cause retention-driven product loss. ![]() Due to their large size, viruses larger than 100 nm also can be retained simply by tight filters. Some viruses are shear sensitive and can be damaged by high shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages through TFF. For example, whereas positive charges increase nucleic acids and HCP capture, diatomaceous earth can retain viruses by adsorption. Based on the size and properties of viral particles, yield could be affected by the clarification method used. Depending on the success criteria for each step, yield can be the main parameter to consider when selecting one option over another for a given step. Yield: In most cases, yield is an off-line measurement conducted at the end of a number of process steps. Other expression systems such as bacteria, yeast, and plant cells also can be used to produce viral particles.Ĭonsiderations on Key Product Quality Criteria and Control Strategies This is gaining interest particularly for producing viral vectors and virus-like particles (VLPs). ![]() Compared with allantoic fluid, however, cell culture harvests are considerably cleaner in terms of solids load and soluble content.Īnother expression method is the baculovirus expression vector system (BEVS) used with insect cells. The proportion of solids in the feed usually is an indicator of purifying challenges ahead (~6–8% mammalian cells or up to 40% in yeast). ![]() Typical contaminants such as host cell DNA, host cell proteins (HCPs), lipids, and bigger particles such as cell debris can be identified. For example, low cell viability can indicate high levels of contaminants released into a feed stream from cell lysis. Based on cell viability - and lysis method selected (e.g., chemical or mechanical), if applicable - the impurity profile in the fluid to be clarified can differ considerably. The composition of these feed streams can vary significantly. Several viral vaccines have moved away from an egg-based process toward the use of live cells (e.g., plant, microbial, avian, or mammalian). It also contains rudimentary tissue compounds from chicken embryos such as feathers, beaks, blood vessels, and/or blood cells. This fluid has a high solids content (>25%, which increases with embryo age) and high mineral and protein content, hence its highly viscous consistency. However, the resulting allantoic fluid harvest (rich in virus particles and cellular debris) is a challenging feed for clarification. In most cases, the virus particles must be kept integral during the clarification step.įor decades, embryonated chicken eggs have been used to produce both human and animal vaccines. We also outline challenges and present current best practices.Ĭonsiderations for Clarification of Viral Vaccines: Type of SubstrateĬomposition, type, and level of contaminants to be removed during clarification mainly depend on the upstream process and expression system used for production. Here we provide a comprehensive overview of different filtration technologies and their application in viral vaccine clarification. Filtration methods include membrane technology (microfiltration operated in normal-flow filtration (NFF) mode, tangential-flow filtration (TFF), or depth filters operated as NFF. Filtration-based technologies have gained prominence in vaccine clarification following the increasing implementation of single-use technologies upstream. Selection of the clarification methodology depends on the type of cells involved, the nature of the virus, and properties of the process fluids. ![]() This is a critical unit operation because it strongly affects product recovery and subsequent downstream purification. Following upstream production and lysis (optional), a clarification step typically is introduced to start the purification process by either centrifugation or filtration. After production, the bulk harvest material is processed to purify a vaccine of interest. They follow a general scheme, starting with production in either an embryonated egg or mammalian/insect cell culture. Viral vaccine manufacturing processes can be templated. Table 1: Classification of viral vaccines
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