From The Stage
← Back to From The Stage
Dr. Afshin Safavi
Posted by Dr. Afshin Safavi BioAgilytix Insight, Biomarkers

Overcoming the Challenges of Critical Reagent and Kit Lot-to-Lot Variability in Support of Clinical Trial Biomarker Studies

Overcoming the Challenges of Critical Reagent and Kit Lot-to-Lot Variability in Support of Clinical Trial Biomarker Studies

Critical reagents are essential components of ligand binding assays (LBA), and include many reagent categories including antibodies, engineered proteins, conjugated proteins, chemically synthesized molecules, complex biologics, and solid-supported reagents. Their unique characteristics are crucial to assay performance, and as such, they require thorough characterization and documentation. Additionally, generation and/or the purchase of critical reagents is a large investment, so setting up proper ways to manage them is extremely important to maintaining the integrity of your assays for years to come. The most important benefit of a critical reagent management system is to assure successful support of analytical and bioanalytical methods and longitudinal studies, enabling the smooth transition of assays between operators and laboratories and timely data collection.

Critical reagents are typically produced via biological processes, and therefore can be inherently prone to variability as different lots are prepared—which can impact the performance of the LBA considerably. Controlling lot-to-lot variability is even more critical when dealing with multiyear studies. The concept of applying correction factors between different lots of assay kits should be discussed and planned before the initiation of the project to generate quality biomarker data so the clinicians and statisticians can make the correct conclusions and decisions. Since there is no standard for such control factors, each lab must set up its own quality control strategy. Labs need to check the expiration dates of kits and have enough for studies that are expected beyond that date. At BioAgilytix, we anticipate more than three months prior to changing kits or to kit expirations. We have conducted studies lasting more than three years and have found that even the same manufacturers can have lot-to-lot variations greater than 200%.

Let’s say you’ve bought your critical reagents and complete the proper fit-for-purpose validation by assessing parameters such as sensitivity, reproducibility, parallelism and relative accuracy, in order to have your validated biomarker assay prepared three to six months before the clinical trial begins. But once the samples start coming in, you receive and run the kits supplied by your vendor—and the results are different. What are the choices now?

As I see it, there are three options when faced with this issue. You can:

1) throw hundreds of thousands of dollars’ worth of kits into the trash (which likely won’t go over well with your organization);
2) return them to the manufacturer (which I have never been able to successfully do); or
3) employ a lot bridging technique, which our team at BioAgilytix has been leveraging for the past 8 years. This chaining or bridging of one lot to another allows us to generate meaningful biomarker data for clients, even when each lot generates slightly different concentration.

I’ll use a case study from one of our clients to explain how our biomarker lot bridging studies work. For their study, they thought it may take one to two years to recruit patients, so we suggested implementing a system that would allow us to bridge from lot 1 to lot 2, since the length of the study would likely require us to use more than one kit lot. In the end, because of patient recruitment challenges, the study ended up running about 3 years, and during this period we used four lots of reagents. Lot bridging studies were performed on these lots using stability samples, and freshly prepared QCs and endogenous QCs (when available) were run on old and new lot kits. The ratios of the QCs’ mean concentrations between old and new lots were examined to determine if there was a meaningful change, and if a correction factor was needed to bridge measurements from kit lot to kit lot. If so, we then used the ratio below to determine the correction factor that a particular pair of lots required.

lot bridging ratio

While our lot bridging method has been very successful, there can be some challenges that come along with biomarker lot bridging. It may result in initial cost increase at the beginning of the project because it will be necessary to purchase additional kits from the new and old lot, and it takes additional time to go through the bridging process. But at the end of the day, kit lot bridging is a needed solution, and using the method we employ or something similar will allow you to produce the highest quality biomarker data across lots.

In conclusion, proper design of lot bridging experiments that measure the effect of using different lots of immunoassay kits are critical to ensuring there is consistent measurement of the biomarker over the course of the study. I recently hosted a webinar for Bioanalysis Zone entitled “Considerations for Evaluation of Accuracy, Parallelism, and Reagent Lot-to-Lot Variability in Single and Multiplex Biomarker Ligand-Binding Assays” in which I cover lot bridging techniques in more detail, along with other biomarker analysis requirements. Click here to view the recording.

Also feel free to reach out to me directly with any questions you may have regarding critical reagent characterization, management, lot bridging, and more.

Share This