: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
CM: Antibodies are great tools. Many exceptionally important research projects would be impossible without antibodies. Thus, researchers need to be able to trust their antibody-based products. At BioLegend, we have always understood that we could never compromise on quality. If an antibody fails in an experiment, far more is lost than that one reagent. No manufacturer should ever find it acceptable to put their client’s research at risk by allowing poorly characterized material to ship to a customer.
Many companies can create an antibody that binds in a single assay to a peptide immunogen or overexpressed recombinant protein. However, such an approach can result in products that give false promises. The fact that something preferentially binds to a target doesn’t mean it will be useful against the native molecule at endogenous levels, or that its preferential affinity for a target will not be swamped by promiscuous off-target binding. The keys to producing a great antibody product are in designing appropriate immunogens, screening for clones in relevant systems, and thorough validation of the clones and materials they produce. This is where the vast majority of effort must be spent. The failure to invest resources in this portion of product development is a key reason some companies fail to achieve a reputation for quality, despite having produced a large collection of antibodies.
If you start with a poor quality clone, then it is very difficult to reproducibly achieve good results. In contrast, if you develop a high-quality clone, control production well, and carefully QC each lot, then you can guarantee reproducible performance against endogenous targets.
: Can you tell us what your company is doing on a routine basis to make sure you are testing and validating the antibodies you manufacture or obtain from other sources?
CM: BioLegend manufactures antibody products at our main facility in San Diego, where we are certified to operate under ISO 9001:2008 and ISO 13485:2003 standards. Our Quality Management System ensures traceability, reproducibility and continuous improvements in manufacturing.
It is very important to us that every product that leaves our facility performs exactly as promised. This starts with validation of all new antibodies against endogenous targets in the applications that will be most relevant to customers. The confirmation of specificity and lack of off-target binding is confirmed with appropriate cellular controls, comparison with any pre-existing antibody clones and appropriate performance in multiple relevant assays. Subsequently, each production lot is QC tested by a variety of biochemical assays, and in the most relevant functional assay for that antibody’s molecular target.
: Do you have any advice, recommendations and/or best practices scientists should follow when selecting and validating an antibody?
CM: When selecting an antibody from a commercial source, there are some basic practices to follow:
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- Source antibodies from a reputable company with a well-established quality system.
- Look for example data generated with real samples.
- Make sure the product you are buying passed a functional QC assay, in addition to the more common biochemical checks (e.g. purity, MW, concentration).
- Carefully compare the demonstrated performance of the antibody in relevant assays to that shown for other available antibodies. If no data is shown, then ask the vendor to provide some. If they can’t readily provide data, then you should be very wary.
- Look for publications that utilized the particular antibody. Companies should try to make these readily available.
- Look at reviews from independent third parties. Many websites, such as Biocompare, SelectScience and AntibodyResource post product reviews by end-users.
When validating an antibody:
- Always follow manufacturer’s recommendations.
- Employ a relevant model system to test the antibody.
- Use ample controls, both for the target (e.g. negative cell line) and for the reagent (e.g. isotype controls that are modified in the same fashion as your antibody).
- Compare to other similar antibodies if available.
- If something looks amiss, contact the supplier’s Technical Service and share your data with their scientists. We consider having expert scientists to assist customers with trouble-shooting or experimental design to be a key part of helping our clients achieve success.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
CM: Developing a minimum set of criteria that an antibody should meet to be considered validated could benefit the industry. There are a large number of players who vary in their level of commitment to quality. If researchers could rely on a certified product meeting a known set of criteria, it would allow buyers an assurance of at least a minimal degree of quality. For those producers and resellers who do not currently meet that standard, it would provide a great incentive to elevate their practices. Having a set of minimum standards is both realistic and implementable.
There has been significant discussion of using recombinant antibodies to create more defined and reproducible products. BioLegend manufactures our mAb products using both traditional hybridoma clones and recombinant methods. A mAb produced off an engineered DNA source is not inherently more defined than one produced from a traditional hybridoma. We often obtain the relevant sequences of our in-house developed hybridoma clones. Whether we choose to express these directly from the hybridoma, from a transfected mammalian cell line, or from a permanent cell line selected to express our recombinant construct is largely decided based on performance and efficiency. In all cases, the peptide sequences of the heavy and light chains we express can be known, but the growth conditions of each production run must still be carefully controlled and monitored for consistency. Recombinant antibody constructs can be useful, but they are not a magic elixir that will guarantee reproducibility.
: What else is your company doing to address reproducibility?
CM: Externally, BioLegend supports a variety of reproducibility and transparency programs, both financially and by contributing products. For example, we have been early supporters of the Resource Identification Initiative, led by Anita Bandrowski, where all of our products are matched to an RRID # (Research Resource Identifier). We’ve also been supporters of the GBSI and are working with them specifically on setting standards for flow cytometry antibodies.
Internally, we have stringent quality systems that monitor each production lot and strive for continuous improvement over our already high standards. During validation, we always look to introduce new technology, new assays and to develop more relevant model systems and controls. The data generated is made publicly available through our website. We have also begun citing contra-indicated applications when we have demonstrated an antibody is not suitable for a particular application. We track and list publications that use our products on our website. Reviews of our products are encouraged and visible through our website. Product stability is continually reviewed via real-time studies in addition to any accelerated stability studies. Our shipping and distribution networks are continually examined to find ways to minimize the chance of delays in-transit to mitigate any associated product issues. Our Technical Service Scientists work to track down the root cause of every potentially problematic inquiry to identify any quality issues that need to be addressed.
BioLegend’s antibody offering is overwhelmingly monoclonal and manufactured in-house. Focusing on mAbs allows us to ensure a long-term reproducible supply. We are also assured that our internally manufactured material will meet criteria to seamlessly pass into our downstream processes that transform antibodies into a variety of different formats and fluorescent conjugates. These modified versions of our mAbs undergo further QC to ensure they function as expected in multicolor panels and multiplex assays.
Author Bio: Craig Monell, Ph.D., BioLegend’s Vice President of Business Operations, has 20+ years’ experience in the Biotechnology industry, including leadership roles in Marketing, Business Development, R&D, and Project Management at PharMingen/BD Biosciences, Bio-Carta, and Stratagene/Agilent Technologies.