: What are the most important factors that a manufacturer should focus on when making an antibody?
RP: Our approach is to focus on the needs of the researcher in order to provide highly specific, rigorously validated antibodies across a broad range of scientific research areas. That translates to the following key manufacturing considerations:
- Specificity—Ensure the performance of the antibody for the intended target. This is accomplished in part by including multiple experimental controls as well as panels of cell lines with known target expression. These results are corroborated using a variety of complementary, orthogonal assays to verify on- and off-target interactions.
- Fit for purpose/functionality—Validation of antibody performance in a particular application including immunohistochemistry, immunofluorescence, chromatin immunoprecipitation, flow cytometry, immunoprecipitation, and western blot.
- Transparency—Providing relevant information, including methods, working dilutions, and data, to support all claims of antibody performance to enable the customer to reproduce results.
- Consistency—Compare each new lot side-by-side to ensure lot-to-lot performs identically to previous validations for a particular target or application. This is especially necessary for the development of polyclonal antibodies. Transparency in revealing this information by any manufacturer is paramount.
: What aspect of your manufacturing process are you most proud of?
RP: The quality of our products is embedded in the scientific culture of Cell Signaling Technology (CST). Founded 20 years ago by research scientists, we’ve maintained our dedication to quality over quantity. We understand how critical our reagents are to the success or failure of an experiment. Ph.D.-level scientists select the targets, determine biological models, design the antigens, develop the testing strategies, refine and evaluate the results, create the protocols, confirm lot-to-lot consistency, and provide technical support across all applications. Our pride is measured not by adherence to budgets or timelines, but by the number of publications that use our products, by the number of manuscripts published from in-house research, and by seeing our own discoveries lead to beneficial diagnostic and therapeutic outcomes. Therefore, our products are gated by our commitment to ensure the release of quality, consistent antibodies.
: How does your company ensure the quality of the antibodies it produces?
RP: Ensuring the quality of our products involves multiple, independent teams in the product development, release, and support process. These scientist-led teams independently test and validate the antibody only when predetermined milestones have been achieved. This process begins at the time of target selection: teams individually review the proposed project, establish testing strategies and determining biologically relevant models for testing, as well as acceptance criteria for each application desired for the antibody product’s use. By following this strategy, no CST antibody product is released without the approval of a development scientist, an application scientist for each approved application, and a group leader who independently reviews all of the data. Then, before the product is available in our catalog, each product must go through an additional round of verification by our strongest critics—the production scientists. They represent the teams that provide technical support, regular quality control testing, and development of all subsequent lots of the same product. If there is any reason to believe that a product cannot be supported both in the short- and long-term, it will not be released. While this extremely cautious approach may result in failed projects, it ensures that only the highest quality, most rigorously validated, consistently produced products make it out our door.
: How do you ensure lot-to-lot consistency with your products?
RP: Each of our antibody products is managed by a team of scientists who are responsible for manufacturing and validating every new lot of antibody before it is released for sale. The process begins by a side-by side performance validation for each new lot against the previous lots for all approved applications. The new lot must perform the same or better than the previous lots before it is approved for release. We also validate each product lot on a regular basis to ensure product quality does not change over time. All of our monoclonal antibodies are manufactured under ISO-9001 guidelines, ensuring consistency and quality.
In addition, CST focuses on making our monoclonal antibodies using recombinant engineering technology, which offers benefits including lot-to-lot consistency, ease of characterization, and decreased production time. Currently, over 95% of our monoclonal antibodies are recombinant, and we’ve expanded this production method to select products made of an optimized mix of multiple recombinant monoclonal clones. Our MultiMabs™ offer all the benefits of a polyclonal without batch-to-batch variation.
Regardless of the production method, all of our antibodies go through a thorough lot-to-lot validation process to ensure that all of our products meet our quality control standards.
: What is your process for validating a given antibody for a specific application?
RP: CST’s application-specific antibody validation begins at the time of target selection. Once a target is identified, each application team evaluates the biology of the target and potential testing models to determine the most appropriate and desired applications to test. These decisions will then determine the antigen and project design. Together with the antigen design team and the application team, the development scientist leading the project is responsible for ensuring the initiation of the most appropriate experiments for each chosen application. All CST antibodies are validated in biological models relevant to the target of interest.
For all applications, CST uses five general categories of validation testing to verify antibody specificity, sensitivity, reactivity, and performance:
- Specificity
- Binary Models & Genetic Inactivation: Using multiple cell lines with known target expression levels and recombinant controls, and also employing siRNA knockdown, knockout models, and other tools
- PTM-specificity: Using appropriate kinase-specific activators, inhibitors, and phosphatases, etc.
- Localization: Assessing subcellular localization and treatment-induced localization
- Biologically Relevant Treatments: Testing cell lines treated with growth factors, cytokines, or chemical activators/inhibitors to knowingly modify target expression
- Tissue Screening: Analyzing multiple normal and diseased tissues to assess performance across a broad spectrum of tissues
- Sensitivity
- Detection of endogenous protein levels
- Optimized application validation and specialized formulations for WB, IHC, IF, flow, and ChIP
- Titration testing in cell and tissue-based arrays
- Consistency
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- Highly reproducible recombinant monoclonal antibody production method to ensure lot-to-lot consistency and stability
- Annual quality control re-validation for all products
- Multiple rounds of independent validation analysis by CST scientists for all product lots
- Methodology
- Product-specific optimized protocols
- Recommendations for optimal dilutions and buffers
- Support
- ○ Top-ranked global technical support—assurance by the scientists who validated the antibody that it will work in your lab
Even using this method, we’re never finished validating our antibodies. As new protocols, methods, models, etc. become available, CST continues to validate its antibodies, both internally and by working with our international community of researcher customers. Our development, application, and production scientists are constantly reading the literature, attending scientific conferences, and speaking with customers. This allows us to understand how our products are used, adapt them to new applications, and enhance our validation data.
: How does your company view the issue of antibody validation? Who is ultimately responsible for ensuring that scientific results are reproducible?
RP: At CST, no one group is solely responsible for ensuring the scientific rigor as there are many stakeholders involved. Suppliers should be held accountable for the products they provide and should be transparent in the source and validation of the products they sell. Vendors should also be held accountable for supporting and continually validating their reagents, as well as working with customers to improve, test, and support their products. Researchers need to remember that antibodies, unlike chemicals used in the lab, are dynamic, meaning sensitive to the context and method in which they are used. Researchers also need to leverage the expertise of the scientists who have developed and tested the antibody and carefully follow established protocols. A manuscript lacking sufficient identification of reagents and materials is, by definition, irreproducible. Therefore, journals need to actively enforce existing policies regarding materials and methods or develop definitive means to identify and describe the use of biological reagents in published research. While tools are being developed to facilitate this process (www.protocols.io, Research Resource Identifiers, etc.), the researchers performing the experiments and writing the papers are ultimately accountable for ensuring their work can be reproduced by others.
: How do you incorporate customer feedback into your processes?
RP: Incorporating customer feedback to continually improve is an ongoing process. We routinely have discussions with colleagues and collaborators from academic institutions around the world and our scientists have active attendance at scientific conferences.
Through feedback submitted via our website, we gather suggestions on what antibodies to make in the future. In addition, we have a third-party product review tool on our website where people can provide reviews on how our products worked in their hands. These customer-written reviews appear unedited on our product pages throughout www.cellsignal.com.
Product consistency is of paramount importance therefore, feedback from scientists like you allows us to ensure that our products will provide repeatable results. We take our customers and product quality seriously.
Author Bio: Roberto Polakiewicz, Ph.D., CST’s Chief Scientific Officer, has been responsible for Product and Technology innovation since 2005. His leadership has created the company's most valuable products and intellectual property. Dr. Polakiewicz has driven the successful implementation of four generations of novel antibody platform technologies critical for CST’s ongoing success. Prior to joining CST, Dr. Polakiewicz received his B.S. in Chemistry and Ph.D. in Molecular Biology from the Hebrew University of Jerusalem and completed postdoctoral studies at the Whitehead Institute for Biomedical Research at M.I.T. Dr. Polakiewicz has published approximately 66 peer-reviewed publications, and is a named inventor on numerous patents and patent applications.