A viral vector is a modified virus engineered to deliver genetic material into cells. These vectors are foundational to modern medical advancements like gene therapy and some vaccines, serving as highly specific delivery vehicles. To function safely and effectively in patients, these vectors must be separated from the complex mixture in which they are produced, a rigorous purification process required before they can be used as a therapeutic.
The Purpose of Viral Vector Purification
The production of viral vectors occurs inside living cells, and when they are collected, the initial mixture contains many unwanted substances. The purification process is designed to remove these contaminants, which fall into two main categories. A primary goal is to eliminate process-related impurities, which include components from the nutrient-rich liquids used to grow the cells and the host cells themselves, such as host cell proteins and DNA. If left in the final product, these materials could trigger an immune reaction in a patient.
Another objective is the removal of product-related impurities. These are viral vectors that are imperfectly formed, such as “empty” viral shells, known as capsids, that do not contain the necessary genetic material. Other product-related impurities can include clumps of vectors, called aggregates. These impurities not only reduce the effectiveness and potency of the treatment but can also cause undesirable immune responses.
Harvesting and Clarification
The process begins in a bioreactor, which contains the cells that have produced the viral vectors. The first step is to get the vectors out of these producer cells through cell lysis, where the cells are broken open using methods like chemical detergents or physical processes like freeze-thawing cycles. The choice of method depends on the specific type of vector and the scale of production.
Once the cells are broken apart, the mixture contains cellular debris, including whole cells and large fragments, that must be cleared away in a step known as clarification. This is often achieved using techniques like centrifugation, which spins the mixture at high speeds to separate the heavier debris from the smaller viral vectors. Another common method is depth filtration, where a filter traps large particles while allowing vectors to pass through.
Chromatography Methods for Separation
Following initial clarification, the core of the purification process relies on liquid chromatography. This method involves passing the liquid containing the viral vectors through a column packed with a specialized material, or resin. Different molecules travel through the column at different speeds based on their physical and chemical properties, allowing them to be separated with high precision.
A highly specific method called affinity chromatography is often a primary step. This technique uses a resin that has a ligand designed to bind only to the desired viral vector. As the mixture passes through, the vectors stick to the resin while impurities are washed away, achieving a significant level of purity in a single step.
Another widely used method is ion-exchange chromatography (IEX), which separates molecules based on their net electrical charge. At a given pH, viral vectors will have a specific surface charge that is different from many remaining impurities, such as host cell proteins or empty capsids. By selecting a resin with an opposite charge, the vectors can be captured while contaminants with the same or no charge flow through. The captured vectors are then released by changing the salt concentration or pH of the buffer.
A final polishing step may involve size-exclusion chromatography (SEC). This method separates molecules based on their physical size. The resin in an SEC column contains pores, and smaller molecules enter these pores, slowing their journey through the column. Larger molecules, like the viral vectors, cannot enter the pores and therefore pass through the column more quickly, removing remaining small protein impurities or aggregates.
Concentration and Final Polish
After the primary impurities have been removed using chromatography, the purified viral vectors are often too diluted to be used as a therapeutic. This is accomplished using a technique called Tangential Flow Filtration (TFF), sometimes referred to as Ultrafiltration/Diafiltration (UF/DF). The TFF system pushes the vector solution parallel to a specialized membrane filter.
This process has two main functions. The first is concentration, where excess water and small buffer molecules are pushed through the membrane’s pores, leaving behind a more concentrated solution of the larger viral vectors. This step is precisely controlled to reach the target therapeutic dose concentration without damaging the vectors.
The second function is buffer exchange, or diafiltration. During this process, the buffer used for purification is gradually replaced with a final formulation buffer. This new buffer is designed to ensure the long-term stability and safety of the viral vector product during storage and upon administration to a patient.
Quality Control and Analytics
Once the purification and concentration processes are complete, the batch of viral vectors must undergo a series of analytical tests to confirm its quality. This quality control step verifies that the purification was successful and the product is safe and effective for clinical use. A number of tests focus on determining the purity of the sample, measuring how much of the product is the correct viral vector compared to any residual impurities.
Analytical methods like the enzyme-linked immunosorbent assay (ELISA) are used to detect and quantify remaining host cell proteins. Other tests confirm the identity of the vector, ensuring it is the exact product that was intended to be manufactured. Further analytics determine the product’s potency and titer. This involves measuring how many of the vectors are functional and capable of delivering their genetic payload, often by assessing the ratio of full to empty capsids.
Finally, safety tests are performed to ensure the product is sterile and free from any harmful contaminants like bacteria or other microorganisms. Only after passing all of these quality control checks can a batch of viral vectors be approved for use.