When it comes to the logistics of clinical trials, it’s time to challenge the status quo, according to Mark Sawicki, chief commercial officer at Cryoport.
Status Quo
The biopharma market is in the early stages of its next transformation. Clinical pipelines are being filled with ever more intricate targets and medicines, including next generation cell and gene therapies. In addition, the diagnostics required to evaluate and progress clinical programs are relying on biomarkers and new assay systems that have increasing sensitivity and complexity. Consequently, logistics costs are rapidly rising to support these industry changes. In fact, the current clinical trial cold-chain logistics market is now estimated to be approximately $3 billion USD annually. Unfortunately, conventional shipping methods including shipping biomarker samples on dry ice are no longer sufficient in many cases. Dr. Dominic Warrino, technical advisor, large molecule sciences at Kansas City Bioanalytical & Biomarker Services (KCAS) has stated that “Approximately 30% of all biomarker samples shipped to KCAS for analysis have experienced temperature excursions.” It is now more critical than ever to look more deeply at the impact of shipping conditions, not only on sample failure, but the impact of the selected transport medium on assay or product performance.
More and more new clinically important biomarkers are being discovered in an ever more challenging array of matrices (CSF, synovial fluid, sweat, tears and whole blood among others), and as such, platforms are being developed and improved to detect biomarkers in these matrices at lower sample volumes and greater dynamic ranges. While each of these new platforms has advantages as well as drawbacks, sample quality is of paramount importance for all to produce reliable sample analysis, informed clinical decisions and effective personalised medicine.
While Quantitative Western Blot has greatly improved in both dynamic range and throughput in recent years, ELISA remains the gold standard for ligand binding assays (LBA) because is well understood, high-throughput and relatively inexpensive. However, it is often subject to deleterious matrix interference effects due to auto conversion of the signal-generating molecule. Electrochemiluminescence (ECL) platforms have a larger dynamic range than standard ELISA’s and also have the benefit of being capable of multiplex analysis. They are, however, still subject to matrix interference effects. In all cases, strict temperature control and maintenance of sample quality can ameliorate some of these effects and increase the dynamic range of a particular assay. As new LBA platforms are developed (immuno-PCR, single molecule counting, proximity ligation assay, Bio-Barcode, etc.) with greater sensitivity and dynamic range, sample quality considerations will become ever more amplified.
LC-MS is a powerful technique for biomarker analysis that is increasing in use due to its ability for high-throughput analysis, detection of low level analytes as well as analytes for which there may not be a good LBA available. Poor sample quality may degrade an analyte, affecting detection levels and % CV within an assay.
Flow cytometry has become an increasingly useful tool for bioanalytical development, and is arguably the most sensitive to sample quality effects. Dead or dying cells, a loss, degradation or down regulation of cell surface markers due to improper sample preparation, storage or transport increases background, signal interference as well as greatly hinders assay performance.
The logistics market has responded to these challenges with new temperature management systems, including phase change materials, vacuum panelled insulators, and actively controlled temperature managed shippers, to move critical materials. These systems do have superior performance characteristics over traditional Styrofoam packaging. Unfortunately, it is likely not enough as a disproportionate number of samples that are critical to evaluating clinical performance are still being moved without a complete understanding of the impact of the transport carrier, packaging, or refrigerant being used. The industry has not conducted a systematic, comprehensive assessment on the impact of these systems and advances under dynamic conditions on the critical materials being moved (namely cell and gene therapies, critical biomarker samples, cell banks, tissue samples, clinical PK/ADA samples, etc.).
Cryoport, in conjunction with our partners, Heat Biologics and KCAS, is conducting a scientific study to evaluate the impact of temperature and transport packaging at deep frozen temperatures on critical biomarkers and cell-therapy cancer vaccines respectively. The study is focusing on the deep frozen space, namely samples typically transported either on dry ice (-80°C) or at liquid nitrogen (-196°C) temperatures. Early results demonstrate that recovery of nearly 30% of all biomarkers tested (8 of 30) were impacted due to shipping on dry ice and assay performance was impacted notably. We also observed significant pH shifts in the samples shipped on dry ice (shifts were observed up to 1.3 points down in the dry ice packages), regardless of container used, a phenomenon observed by others in the recent past to have an impact on sample integrity. Dynamic temperature mapping experiments also indicated a significant level of thermal variability in both dry ice packages utilised versus cryogenic shipping. These early results substantiate the concern the bioanalytical industry has expressed and support the call for a more comprehensive assessment in the coming months and years.
In addition to sub-optimal (and in some herein observed samples, statistically disqualifying) biomarker recovery and assay performance due to either temperature or pH shifts, one also needs to consider the impact of the transportation itself. As part of the study, we looked at the orientation and handling of the packages in transit. Out of the 33 packages shipped, 32 had multiple orientation events and the average time spent in a mis-oriented position averaged between 10-15%. The type of package was not consequential as all package types incurred such handling issues and the handling issues occurred with both integrator and specialty courier managed packages. Mis-orientation is important as it impacts both dry-ice sublimation rates and nitrogen evaporation rates, thus reducing the overall hold time of the package. In the case of dry ice shipping, increased dry ice sublimation rate increases the amount of acidic carbonic gas and may accelerate pH shifts in the samples. In addition, package handling can damage the packaging integrity, introduce shear related stress on the sample being moved, and subject the sample to increased temperature volatility.
As per the study conducted, it is believed that all of these factors (temperature, pH, orientation, dry ice vs. LN2) play a role in the quality of biomarker and/or clinical samples and may not only impact the quality of the data generated for evaluation of more than 64,900 studies active as of the time of this submission, but will also impact release of pharmaceutical drug substance, drug product, and final dosage products which all rely on analytical assays for verifying product quality and performance.
Our study makes clear that packaging and logistics decisions directly impact sample quality and bioanalytical assay performance. Based on the early results we have observed, shipping biomarker samples at cryogenic temperatures does in fact reduce thermal variability, eliminates the risk of pH shifts, and improves overall assay performance in the biomarker panel tested. It is important to note that for all biomarkers tested the mean recovery rate and %CV of the samples shipped cryogenically were comparable to control, indicating that shipping biomarker samples cryogenically is safe for all biomarker samples, and for 30%, is critical for optimal recovery and assay performance.
We encourage the industry to take a step back and critically evaluate whether or not clinical efficacy, as well as bioanalytical or manufacturing issues may in fact be due to logistics choices. It is time for a wide-ranging industry wide assessment on the most appropriate logistics methodology and standards for maintaining appropriate sample integrity, activity, and performance.