Frontage Labs

Q+A on Critical Reagents Usage in Bioanalytical Labs

Santosh Shah

Dr. Santosh Shah

Director, Biologics, Frontage Laboratories

Dr. Santosh Shah is the Subject Matter Expert and Director of Biologics, at Frontage Laboratories. He has been providing scientific leadership and has been contributing to the development of the Global Biologics Strategy. He works as a liaison between business development, operations, and various other departments. He is also involved with communicating, auditing, and presenting bioassay capabilities (PK, ADA, NAb, Biomarker) to the pharma sponsors. He has been leading the automation development team at Frontage.   Dr. Shah discovered several highly potent, copper-mediated, drug compounds against M. tuberculosis and S. aureus during his Postdoctoral Fellowship at the University of Alabama at Birmingham. During his Ph.D. at the University of Arizona, he dedicated himself to understanding the structure-function relationships of DNA nucleases.

Critical reagents for ligand binding assays (LBA) are essential components of bioanalytical testing. These include but are not limited to antibodies used as positive controls, capture, or detection reagents; peptides as drug targets; reference standards as test analytes for pharmacokinetics; positive controls for immunogenicity studies and matrices.

Critical reagents are crucial for assay performance and data comparability across different laboratories and countries since they directly impact the results of an assay. Thus, they need to be well characterized and validated for the proper execution of preclinical and clinical studies. Critical reagent stocks must also be maintained, and their sources recorded. Below is a snapshot of some of the key questions asked on the webinar, General Practices for Critical Reagents Usage in Bioanalytical Labs.

In the following Q+A, Dr. Santosh Shah, Director of Biologics, answers key questions on critical reagent usage in bioanalytical laboratories.

What approach would you use to bridge reference standards if you want to compare head-to-head, old, and new curves and full complement of quality controls (QC): LLOQ, LQC, MQC, HQC, ULOQ?

We set up two sets of plate controls (i.e., one set = one set of standards and two sets of QCs) each prepared with two different lots separately, and check the following results:

For each set, if standards and QCs meet the acceptance criteria.

If one lot of prepared QC meets the acceptance criteria in comparison to another lot of prepared standards.

Preparing from an old and new batch of reference standards will not fit on a 96-well plate, if there is the need to include three replicates of each. Is just running one or two replicates a valid approach so all five levels of QCs can be tested?

Our most common practice is to use two replicates. If three replicates are required, we use two plates to complete the comparison of reference standards.

Do you have a standard expiry assigned to different types of critical reagents, since often the stability is not known initially? Would you be willing to share your approach for being able to assign the expiry or retest/recertification dates initially?

For critical reagents acquired from external sources, we use expiry dates from certificates of analysis (COAs) provided by the sponsor/vendor. For critical reagents prepared in-house, we usually follow our standard operating procedure (SOP) based on the type of reagent, e.g., for an antibody-based critical reagent, we assign a 1-year expiry and retest annually.

How do you apply the correction factor, as you see >20% QC bias between using two different lots of reagents? In other words, will the correction factor be applied to assay modification or study data?

The correction factor is usually used when bridging sample analysis kits, in which standards and buffers are included for standard calibrators and QCs preparation. From kit to kit, the buffer and standard material are different to a certain extent and the overall comparison in QCs will be greater than individual standard material lots. The correction factor will be applied to the study data, not the assay.

Can you speak on extending expiration dates via retesting?

We perform one run with one set of standards and two sets of QCs (HQC, MQC, and LQC) loaded to plates in duplicate. If the run passes, the expiration may be extended for a time equal to the original time interval.

What is the purpose of BIO-006 Change Control Procedure?

BIO-006 Change Control Procedure SOP provides a procedure for managing document and equipment changes at Frontage Laboratories. It outlines the method of documenting and controlling changes to issued documents and equipment, as well as tracking changes.

What should the re-testing frequency for in-house conjugated reagents, e.g., biotinylated drug or digoxin drug, be?

We usually retest once a year since the labeled reagents tend to become aggregated, leading to a higher signal over time. This change is more detrimental to ADA studies in which the raw data are evaluated for positiveness and titer of the ADA confirmed. We would rather re-label reagents than re-testing the regents.

Frontage’s biologics teams have nearly 15 years of experience in complex drug development and large molecule applications throughout its evolution in product development. We have handled projects for peptides, proteins, monoclonal antibodies, bispecific antibodies, biosimilars, oligonucleotides, biomarkers, and antibody-drug conjugates. Contact our sales team for your biologics projects.

Note: This feature includes the webinar, panel discussion, and Q+A, and was done in association with Bioanalysis Zone.

Gyrolab: The Platform

Gyros Protein Technologies AB developed Gyrolab platforms using “reproducible nanoliter microfluidics and sequential CD processing” for efficient immunoassay runs. The first immunoassay system for nanoliter-scale protein quantification was released in 2003 at Drug Development Tool (DDT), Boston. In 2009, Gyrolab xP launched a new software designed for 21 CFR part 11 compliance and extended validation support. It miniaturizes immunoassay and integrates reagents (capture and detection antibody), sample addition, and CD washing into a single machine. For pharmacokinetic (PK) studies, it has brought advantages like broader dynamic ranges, shorter time to results, reduced sample and reagent volumes, and automated programs.

Gyrolab Design

The Gyrolab system works with its proprietary CD technology engineered with highly reproducible nanoliter microfluidics. Each CD has 96/112 channels depending on the CD type and each channel has an individual sample addition inlet. Through precise, automated control of CD spin, capillary forces steer liquid flow through nanoliter-scale microfluidic structures in each CD channel and form the sandwich format. Fluorescence data is collected from the assay and analyzed using the Gyrolab Evaluator software to obtain final protein concentrations.

Challenges and Advantages of Gyrolab 

Different from MSD and ELISA platforms, which use disposal tips to transfer reagents and samples, Gyrolab uses needles to transfer samples. Because of the instrument’s design, carryover contamination checks are necessary to conduct during method validation. For optimal use of Gyrolab, Frontage’s bioanalytical team shows how carryover contamination checks can be performed to ensure smooth operation.

Unlike the MSD and ELISA platforms which can handle multiple plates parallelly, Gyrolab processes CDs in a sequential manner. One CD run can take 1 to 2 hours, and the last CD needs to wait about 4 to 8 hours on the machine to be processed. In this case, samples after MRD benchtop stability need to be established to make sure samples are stable on Gyrolab while waiting for analysis. For optimal use of Gyrolab, Frontage has performed “After MRD (minimum required dilution)” stability testing to mimic the delay in loading standards.

No doubt, the Gyrolab platform has several advantages over ELISA or MSD platforms. The broader standard curve range and the proprietary CD technology which uses affinity flow-through assay reduce the background noise significantly and increase the sensitivity. It runs in a shorter amount of time and can utilize smaller sample and reagent volumes, saving critical reagents. An ideal choice for preclinical drug development animal assays, which have limited sample volume, it is also suitable for clinical projects with many samples that need to be analyzed in a short time.

Using Gyrolab for Your Immunoassays

With an increased need for high-throughput immunoassays using precious samples and uncompromised data quality in a limited time, choosing a CRO that can build quality assays and deliver robust data is critical. Frontage’s Gyrolab team has deep expertise in analyzing any peptide, protein, or antibody, and with method validation using the Gyrolab platform under GLP or Clinical environments.

Preclinical Drug Screening Using Gyrolab

Author’s Note: This article is written with support from Zhongqiang Qiu, Ph.D. from Biologics Service at Frontage Laboratories.