Automated Frozen Sample Aliquotting: Improving Bioanalytical Assay Results for Labile Molecules

Automated frozen sample aliquotting technology is now available from CryoXtract. Frozen aliquotting enables bioanalytical scientists to obtain quantitative aliquots from study samples while still frozen at -80^oC. Instead of using a traditional pipette or liquid handling workstation to transfer thawed aliquots, a 3mm hollow coring probe is used to transfer cylindrical frozen core aliquots from standard cryotubes for downstream analysis. By avoiding damaging freeze/thaw cycles and maintaining the ‘cold chain’ during sampling, PK plasma samples (for example) can be re-analyzed many times without fear of analyte degradation or precipitation of the matrix components, which is often observed in thawed samples. Incomplete or inconsistent mixing of thawed samples is known to be a chronic source of concern and error in bioanalytical labs and is even the root cause of some failed incurred sample reproducibility (ISR) studies. Frozen sample aliquotting addresses both the stability and mixing concerns.

Frozen sample aliquotting can solve most of the stability challenges that plague bioanalytical scientists in both drug discovery and regulated GLP bioanalytical labs. The technology is equally applicable to LCMS assays, ligand binding assays (e.g. ELISA), and hybrid analytical assays. Most therapeutic drugs, their metabolites, and endogenous proteins and biomarkers are susceptible to both thermal hydrolysis and enzymatic degradations. Frozen core aliquotting helps ensure the most accurate measurement of analytes in biological samples. The technique is especially noteworthy when working with and measuring unstable compounds. In most cases, frozen aliquotting can replace the current practice of using pH buffers and/or stabilizing reagents such as costly – and sometime assay interfering – enzyme inhibitors. Because these pre-analytical practices are known to be quite error prone, costly, and time consuming, frozen sample aliquotting is also a great way to simplify the sample collection process. In the most extreme cases, frozen aliquotting could be used to further enhance the stability of highly unstable compounds such as; ester pro-drugs, endogenous peptide hormones and auto-oxidizing natural products.

The application was first evaluated in GlaxoSmithKline’s DMPK bioanalytical labs in Research Triangle Park, NC. This talk will share the detailed system performance data of CryoXtract’s CXT 750 automated workstation and the significantly enhanced stability and reproducibility data seen when analyzing a mixture of unstable small drugs and peptides by HPLC-MS/MS, in EDTA plasma, over 5 consecutive days. Specifically, the system performance was initially evaluated in terms of:

1. Precision and accuracy. Precision and accuracy of replicate aliquots in the typical LCMS sample volume range of 50 to 100 uL aliquot size, measured both gravimetrically and using an establish HPLC-MS/MS assay. Reproducibility (as % CV) and accuracy (as % bias) results were all within the acceptable +/- 15% range.
2. Carry-over. Carry-over was assessed by coring and assaying replicate blank plasma samples immediately after the coring/aliquotting of QC samples at twice the upper concentration limit of the LCMS assay used. No carry-over was observed (< 0.02%).DIV>
3. Uniformity of target analyte distribution. Core aliquots of QC samples were taken from both the center and peripheral of cryotubes, as well as from different vertical depths. No significant differences in analyte concentration were observed between those core aliquots, indicating that the small drug molecule was evenly distributed in the EDTA plasma samples which were initially frozen and stored at -20^oC.

The stability study looked at a mixture of a dozen unstable small drug molecules and peptides. The mixture was spiked into both rat and human plasma as batch QC test samples. Quantitative aliquots (n=3) of each were assayed immediately to establish the LCMS reference response of each analyte. The remaining volume was quickly separated into individual cryotubes and frozen at -80^oC. Two sets of each QCs were then re-assayed (in replicates of n=3) for 4 additional and consecutive days. One set was re-assayed each day using traditional freeze/thaw liquid aliquotting, the other set was aliquotted each day on the CXT 750 at -80^oC. All compounds were seen to remain stable through 4 additional re-assay cycles on the CXT 750. Conversely, most of the analytes were observed to degrade to some extent over the course of 4 additional freeze/thaw cycles. The extent of degradation varied from no observed degradation (photo-sensitive compounds) after 4 cycles, to >95% degradation after the very first thaw cycle (cistracurium – a short acting neural-blocking anesthetic agent).

The talk will conclude with a discussion on current studies and evaluations looking at establishing best practice recommendations for the collection and freezing of pre-clinical and clinical samples, to ensure the best possible uniformity of analyte distribution for frozen aliquotting. CryoXtract is currently looking at how; freeze temperature, matrix type, anti-coagulant, and analyte type (small molecules to mAbs) might affect the uniformity of an analyte’s distribution in frozen samples.

Speaker

Glenn Smith, Independent Scientific Consultant for CryoXtract, and Former Section Manager of the DMPK bioanalytical labs at GlaxoSmithKline Inc. in Research Triangle Park, NC

Glenn Smith has worked as an independent scientific consultant for CryoXtract since April 2015, and prior to that was Section Manager of DMPK bioanalytical labs at GlaxoSmithKline Inc. in Research Triangle Park, NC. Smith has over 25 years experience in bioanalysis and laboratory automation. He has worked in the CRO and Pharma industries and is considered an expert in aspects of quantitative bioanalysis including; assay development & validation, sample preparation, HPLC & LCMS, GLP & regulatory, instrumentation & lab automation, sub-cellular drug analysis, and lab management & process improvement. In 1999, he received the ‘’Pioneer in Laboratory Robotics’’ award at the International Symposium on Lab Automation and Robotics (ISLAR) for his work at Glaxo Inc. & GlaxoWellcome, Inc. His work led to modern-day automated high throughput parallel (96 well plate based) bioanalytical sample preparation. He’s authored and contributed to over 40 scientific publications and presented at over 20 scientific meetings.

Message Presenter