Webinar Q&A follow up: utilizing LBA and critical reagents for drug development
Thank you to those who attended our Panel discussion titled ‘utilizing LBA and critical reagents for drug development’, in association with Frontage Laboratories (PA, USA). Below are responses to the questions posed by our audience during the live event. We hope this is a useful resource and thank those who submitted these thoughtful questions.
1. What are the challenges associated with using commercial kit reagents for a pharmacokinetic (PK) assay?
- Ensuring that the provided reference standard will perform equivalent with the drug product in clinical studies.
- Calibration standards need to be prepared in-house if the kit does not provide enough standards, including LLOQ and ULOQ.
- Quality controls (QCs) need to be established according to the study sample concentration range.
- The buffer provided in the kit may significantly differ from study sample matrix.
- A new lot of kit may perform with a high variability and comparability to previous lots.
- Multiple plates may show high variation in coefficient of variation (CV) and relative error (RE) of QCs.
2. Do you have a suggestion for calculating cut point in simple way?
As per FDA guidelines, the screening cut point factor (S-CPF) is calculated based on 5% false positive rate.
S-CPF = “Mean + 1.645× SD” (if data normally distributed); or “95th percentile” (if data not normally distributed)
The %Inhibition and confirmatory cut point (CCP) is calculated using a 1% false positive rate.
%Inhibition = 100 × (ECL without drug – ECL with drug) / ECL without drug
Confirmatory Cut point (CCP) = “Mean %Inhibition + 2.33 * SD %Inhibition” (if data normally distributed); or “99th Percentile” (if data not normally distributed)
Titration cut point factor (T-CPF) is calculated based on 0.1% false positive rate.
T-CPF = “Mean + 3.09 × SD” (if data normally distributed); or “99.9th percentile” (if data not normally distributed)
All the calculations can be done with statistical software or Excel.
3. Regarding reagents, do you have any suggestions for resolving matrix interference in ligand binding biomarker assays, especially for parallelism?
- Increasing minimum required dilution (MRD) can weaken matrix interference to some extent.
- Adjusting LLOQ to another level to reduce the impact of the matrix interference.
- Develop a new method, avoiding the potential effect of endogenous binding protein that may affect parallelism substantially.
4. How do you foresee your current Laboratory Information Management System (LIMS) evolving in order to meet the needs of critical reagents, from creation, labelling, usage and overall tracking?
LIMS may be upgraded so that it can dynamically monitor commercially available bioavailability / bioequivalence (BA/BE) or biosimilar drug related kits, critical reagent pairs, available vendor capacities and new technologies applied to customized product options. LIMS can be customized to fit the needs of different stages of method development, validation and sample analysis. LIMS may evolve to integrate other portals such as QB, EWB and other study management related software to track all study related critical reagent lifecycle, inventory and re-stocking schedule.
5. When diluting samples to prevent interference, is the dilution performed with a control matrix, stripped matrix or a reagent?
Usually a control matrix or blank matrix is used for dilution. However, stripped matrix can also be used if it is available from a vendor, or if the stripping method works properly. If interfering molecules cannot be removed physically, a surrogate matrix may be used.
6. What is a typical lead time for creating critical reagents when outsourced? Does this depend on the type of labels being used?
For polyclonal antibodies (pAbs), lead time is about 2-4 months depending on the quantity and shipment conditions; for monoclonal antibodies (mAbs), it could take 6 months or longer. The lead time is not directly related to label types applied to the reagents.
7. What range of lead times for reagent prep can be expected, considering the different types of reagents that can be prepared? What are the longest/shortest expected times?
Lead times are variable dependent on reagent type, vendor and timing. Most of them take several months to a year.
8. Generally speaking, how is the reproducibility/reliability of commercial primary antibodies/kits? Do we need to develop internal antibodies?
For the routine biomarker kits, the performance is very consistent. The commercial primary antibodies vary significantly, depending on whether the analytes are common or unique. If a kit or commercial Ab does not work well to fulfil the requirements of the study sample analysis, internal Ab development may be initiated.
9. Typically, how long does it take to develop LBA methods using commercial reagents vs generating reagents internally?
It takes 3-4 weeks to develop LBA methods using commercial reagents. However, it takes significantly longer time to develop methods by generating reagents internally.
10. What are the significant drawbacks/risks of local analysis Vs. analysis post-long-distance shipping? What are the future solutions?
The data submission timeline and cost will be different between the two analysis ways. The possible solutions are: (1) shipping samples to a local CRO with a confirmed technical team and trusted QC and QA; (2) locally operating clinical studies; (3) outsourcing clinical studies to local CROs.
11. Is Quanterix’s Simoa also a preferred platform?
Quanterix’s Simoa is preferred depending on the assay sensitivity requirement. Since Meso Scale Discovery (MSD) and ELISA-based assays cannot reach a pg/mL sensitivity, SIMOA is a preferred platform to attain such sensitivity.
12. Can you introduce the application of hybrid LBA/LC-MS for ADA assays?
The widely used LBA-based ADA assessment is a qualitative assay with a quasi-quantitative titer analysis. However, the hybrid LBA/LC–MS is a semi-quantitative assay which has unique features such as direct ADA measurement, good detection, specificity and powerful multiplexing/isotyping capabilities.
13. Other than avoiding the buffer pH being close to isoelectric point (PI) of the protein, what else can we do to improve stability?
In order to improve stability, 0.1% bovine serum albumin (BSA) can be added to the manipulated reagents, e.g., biotin or sulfo-tag labelled drug, capture or detection antibodies or molecules. For original critical reagents, they can be left in the original formulation. Prepare smaller one-time use aliquots and avoid multiple rounds of freezing/thawing. For lyophilized reagents, reconstitute it in the recommended buffer before using.
14. Can you explain how increasing assay incubation time and reagent concentration helps to eliminate/reduce matrix interference?
The quality and quantity of critical reagents play a major role in LBAs. Increasing assay incubation time and reagent concentration will push the equilibrium towards the desired complex formation and compete out any interfering components in the matrix, thus minimizing unnecessary matrix interference.
15. In some areas of the field, critical reagent labeling is being automated. Do you have any thoughts on the consistency and quality of reagent labeling by automation?
For a bulk reagent labeling, automation used for labeling is a preferred option because manual labeling Dye-to-Antibody Ratio (DAR) may vary from lot-to-lot.
16. To generate antibodies for a smaller molecules such as peptides, what’s the best strategy?
Conjugating peptides with 1000-2000 kDa with keyhole limpet hemocyanin (KLH) or BSA is a regular strategy. Utilizing nanobody technology might be the best strategy.
17. What is the preferred buffer to store labeled reagents?
The original formulation is the preferred buffer for storage of labeled reagents. The preference will also vary based on the type of reagents and their PI. Labeled reagents could be categorized based on their similarities and similar type of buffer for each category could be used.
18. What are the advantages of hybrid LBA/LC-MS platforms?
The hybrid LBA/LC-MS platform utilizes the enrichment step usually carried out in LBA format —beads or columns— and the analysis step carried out by LC-MS for improved selectivity and sensitivity. There are multiple advantages to this hybrid method; LC-MS does not require multiple labeled reagents like LBA platforms, thus avoiding issues brought about by the labeled reagents, such as lot-to-lot variability and reagent generation time. Significant robustness testing is not needed for LC-MS since it does not have multiple variables as in LBA platforms.
19. What system is being used for tracking reagents at Frontage?
The Critical Reagent Management Team at Frontage utilizes a Bioanalytical Reagent System Tracker and Electronic Workbook to maintain updated reagent inventory and notification. An annual report provided by the sponsor also includes a summary of all the reagents used and predication of the upcoming year’s estimation. These allow us to plan ahead in procuring and storing the required reagents.
20. What are the main challenges regarding critical reagents when working with peptides in contrast to mAbs?
Stability of peptides and method robustness are the main challenges since the peptide sometimes cannot form a stable structure with other reagents.
21. Are you familiar with the ELLA (Protein Simple) platform? How popular do you estimate it will be in 10 years’ time?
Since the sample capacity, method flexibility and cost are the main concerns for this platform, its popularity will likely be limited for next 10 years.
22. How can we generate or identify stability in critical reagents generated in-house?
Based on the performance: if the critical reagents generate a consistent signal over time, the stability of the reagent should be good enough for the given assay. In addition, using a simple polyacrylamide gel electrophoresis (PAGE) gel will detect the integrity of the critical reagent. If it is client generated, request for the critical reagent to be tested and a new certificate of analysis (CoA) issued to ensure stability.
23. How often do you use automation in PK or ADA assays? What are the critical factors method developers should consider when using automation to develop these kinds of assays?
Automation can be used more frequently for PK assays than for ADA assays given the challenges to setup ADA assays. A critical factor for automation method development is to standardize methods so fewer modifications need to be done while developing new methods. The methods should be simple, streamlined, error proof and require limited user input. Use the correct liquid class, tips and labware for better performance. Other factors to consider include efficiency, precision, reproducibility, study size, reagent consumption and the quality of data generated.
24. How important is it to characterize the labeled reagents? Based on your experience, do you see a need to run labeled reagents through size exclusion columns?
It is essential that a well-characterized labeled reagent is used in regulated studies to have confidence in the method development. Information from CoA and DAR can aid in development and optimization of methods efficiently.
Size exclusion columns can be used to concentrate the labeled reagent and remove the excess non-labeled reagent, thus minimizing the background noise. LC-MS can be used to aid in characterization of labeled reagents.
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