Cell and gene therapy holds immense potential to revolutionize the field of medicine by addressing the root genetic causes of diseases or empowering cells to fight against them. This approach has already achieved remarkable successes in healthcare, including the treatment of rare eye diseases, sickle cell disease, and various cancers.

One of the key tools used in cell and gene therapy are viral vectors, which are specialized vehicles that transport genetic material to target cells. Viral vectors have a wide range of applications, from gene therapy to vaccine development. However, before they can be safely used in humans, they must go through a rigorous purification process to eliminate impurities and ensure the safety and efficacy of the final product.

The purification process for viral vector production involves multiple steps that vary depending on the type of vector and production system used. Along the way, quality control tests are performed to ensure that the final product is safe and effective. These tests include assessing vector potency, identity, and purity, as well as checking for the presence of contaminants such as adventitious viruses or endotoxins.

Despite the great promise of cell and gene therapy, there is a significant bottleneck in the production of viral vectors due to the explosive growth of this field. The current manufacturing capacity for viral vectors falls far short of the demand, which has resulted in a challenge for manufacturers to keep up with the increasing regulatory scrutiny and product characterization requirements. This situation has put pressure on manufacturers to adopt rapid, robust technologies for viral vector analytics and characterization and to conduct rigorous quality control assays to speed up the development of gene therapies.

Importance of Immunoassays in Gene Therapy Development and Manufacturing

Immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), play a crucial role in the development and manufacturing of cell and gene therapies. These assays are used to evaluate viral vector titer and assess immunogenicity responses to gene therapy treatment. For example, titer analysis is commonly used for adeno-associated virus (AAV) and lentivirus vectors, which are two of the most commonly used vehicles for introducing genetic therapies into mammalian cells.

AAV has low immunogenicity and toxicity, making it one of the most promising viral vectors used for delivery of genetic material in gene therapy. However, developing and producing AAV vectors at an industrial scale requires an efficient method for accurately quantifying viral capsids in both upstream and downstream bioprocessing. Quantifying functional viral titer for lentiviral or AAV vector production can be time-consuming and costly. Therefore, it is essential to respond quickly and adjust culture or bioprocess conditions to optimize vector titer, which quality checked through ELISAs.

During the manufacturing and purification process of viral vectors, impurities from host cell proteins (HCPs) of HEK 293 or CHO cells, commonly used for expressing viral vectors, can potentially contaminate the final product. To avoid unfavorable toxic or immunological reactions, it is preferable to minimize HCP impurities to its minimum during vector manufacturing. HCP measurement is a critical regulatory requirement, and having an open platform with the flexibility to measure HCPs from new expression host systems or utilizing in-house developed assays is essential. Due to its simplicity and high sensitivity, ELISA has also become the standard technique for measuring HCPs.

Considerations for Optimizing ELISA Assay Performance in Cell and Gene Therapy Manufacturing

ELISA plays a critical role in cell and gene therapy, and selecting the right immunoassay platform can increase productivity by streamlining workflows. However, there is an essential step in every ELISA workflow that cannot be neglected and can create a bottleneck within the workflow: the washing steps. It is essential to reduce background signal related to unbound conjugated antibodies. Leaving any unbound material in the wells can increase background noise, reducing the sensitivity of the assay and the quality of the data obtained. Inadequate washing can lead to poor results.

Several parameters affect the effectiveness of wash steps in ELISAs, and wash volume is one of the most critical ones. To minimize high background or variation, it’s essential to adjust the wash volume to at least match, or preferably exceed, the coating volume of the well. Insufficient washing volumes leave parts of the assay surface unwashed and significantly increase the background signal. It’s crucial to ensure that the washing volume is equal in all wells. To minimize well-to-well variability, it’s also essential to perform sample pipetting and washing accurately and consistently. The use of multichannel or automated pipetting and wash systems, such as microplate washers, may improve the results. However, residual volume per well after liquid removal can vary based on the methods or microplate washer used. A lower residual volume implies more efficient washing, eliminating unbound material rapidly. However, there is a constraint around the type or hardware used, making it difficult to go below 10 µl of residual volume per well. Therefore, the plate would need to be washed multiple times to achieve optimal washing, consuming therefore more washing buffer, but also more consumables for washing methods relying on pipette tips.

For example, using the same pipette tip or any washer that comes in contact with the liquid for aspiration can propagate material from plate to plate. This can compromise the specificity and sensitivity of the assay, leading to false-positive or false-negative results. To prevent cross-contamination, it’s essential to use separate tips and avoid contact between the liquid and the washer or any other materials that might be a source of contamination.

Given the extensive validation, metrics, limits, and acceptance criteria used to evaluate ELISA assay results, it is understandable that many laboratories are reluctant to change their validated workflows. However, there are washing solutions available that can reduce washing cycles by up to 3-fold while enhancing the overall assay performance. Adopting these solutions can increase productivity and streamline workflows in high-pressure manufacturing environments.

Revolutionize Your Immunoassays with CYTENA’s C.WASH Microplate Washer

CYTENA’s C.WASH is an innovative microplate washer designed to automate and streamline the washing of microtiter plates, making it an ideal solution for labs focused on immunoassays. This state-of-the-art instrument is designed to enhance data quality, improve reproducibility, and reduce time and cost in immunoassay workflows.

C.WASH is a non-contact strip microplate washer. C.WASH uses centrifugal forces to remove liquids from well plates in seconds, eliminating the risk of carryover and cross contamination. With a residual volume of under 100nl, the instrument drastically reduces the number of washing cycles required hence lowering the background level. This leads to better and finer detection of the target analytes.

C.WASH can operate up to four liquids with auto-priming features and minimal internal dead volume, providing optimal flexibility and precision in your immunoassay workflows. Additionally, the instrument eliminates the need for pipette tips, which can lower facility spending on consumables.

Waste management also benefits from C.WASH’s efficient washing cycle, which requires only two cycles to achieve 100% washing efficiency (performed under 2 minutes), reducing the volume of washing buffer necessary by 67% compared to conventional methods.  With C.WASH, labs can improve their research efficiency, accuracy, and reproducibility while reducing costs and improving waste management.

Finally, due to its SiLa2 compliance, integrating C.WASH into an existing automated platform is effortless and cost-effective, allowing for seamless integration with other systems and software services.

C.WASH system offers a highly efficient solution for manufacturing vectors for cell and gene therapy. With its state-of-the-art technology, C.WASH reduces hands-on time and enhances productivity, enabling researchers to optimize their workflows and achieve faster results. Being SiLA2 compliant, C.WASH provides a cost-effective solution for laboratories seeking to improve their operations. Additionally, the C.WASH can help labs reduce their carbon footprint and lower consumable costs associated to pipette tips consumption.

By using C.WASH, laboratories can increase their capacity to produce high-quality vectors for cell and gene therapy, ultimately contributing to the advancement of this rapidly evolving field.

For more information, please visit: C.WASH.

About the Author

Dr. Thomas Clapes is a Molecular and Cellular Biologist with a PhD from Erasmus University Rotterdam (2014). He gained valuable postdoctoral experience at the Max Planck Institute of Immunobiology and Epigenetics in Freiburg, Germany, before becoming a Global Product Manager at Cytena GmbH in 2021. Dr. Clapes is dedicated to driving scientific progress by developing innovative products and laboratory instruments tailored to meet the specific needs of researchers worldwide.