FDA Quantitative Immuno PCR Detection Instruction Manual

FDA Quantitative Immuno PCR Detection

Product Information

The product is a quantitative immuno-PCR assay designed for the detection of plasma IP-10 levels over time. It is specifically developed as a candidate pharmacodynamic biomarker of IFN-1 biologics, such as interferon beta-1a (IFN- 1a) and pegylated IFN-1a
(pegIFN-1a). The assay is intended to provide clinical pharmacodynamic similarity data to support the approval of biosimilars and guide subsequent clinical testing.

The assay utilizes proteomic analysis of plasma samples to identify IP-10, an interferon-gamma-inducible protein-10, as the target biomarker. It offers a quantitative measurement of IP-10 levels in plasma samples, allowing for the evaluation of its concentration over time.

Key Features:

• Quantitative immuno-PCR assay
• Target biomarker: Interferon-gamma-inducible protein-10 (IP-10)
• Designed for IFN-1 biologics (IFN-1a and pegIFN-1a)
• Provides clinical pharmacodynamic similarity data
• Supports approval of biosimilars
• Guides subsequent clinical testing

Product Usage Instructions

Please follow these instructions for using the quantitative immuno-PCR assay:

1. Collect plasma samples from the subjects or patients following standard protocols.
2. Prepare the assay reagents according to the provided instructions.
3. Thaw the plasma samples and ensure they are at room temperature before beginning the assay.
4. Label the microplate wells according to the sample identification.
5. Add the appropriate volume of each plasma sample to the designated wells.
6. Add the assay reagents to each well, following the specified volumes and mixing instructions.
7. Seal the microplate and gently shake to ensure proper mixing of the samples and reagents.
8. Incubate the microplate at the recommended temperature and duration.
9. After incubation, wash the microplate wells thoroughly to remove any unbound substances.
10. Add the detection reagents to each well, following the specified volumes and mixing instructions.
11. Seal the microplate and gently shake to ensure proper mixing of the detection reagents.
12. Incubate the microplate again at the recommended temperature and duration.
13. After incubation, wash the microplate wells thoroughly to remove any unbound detection reagents.
14. Add the substrate solution to each well, following the specified volumes and mixing instructions.
15. Incubate the microplate for the final time at the recommended temperature and duration.
16. Stop the reaction by adding the stop solution to each well.
17. Measure the absorbance or fluorescence of each well using a compatible plate reader.
18. Calculate the IP-10 concentration in each plasma sample based on the provided standard curve or calibration equation.

Note: It is recommended to carefully read and follow the detailed instructions provided with the product for accurate and reliable results. Proper handling and storage of reagents and samples are crucial for assay performance.

Quantitative Immuno-PCR Detection of Plasma IP-10 Levels Over Time:
Candidate Pharmacodynamic Biomarker of INFβ-1 Biologics
Deepti P. Samarth1, Lakshmi Manasa S. Chekka1, Barry Rosenzweig1, Jeffry Florian1, David G. Strauss1, Paula L. Hyland1Division of Applied Regulatory Sciences, Office of Clinical Pharmacology (OCP) / Office of Translational Sciences (OTS) / Centerfor Drug Evaluation and Research (CDER), U.S. FDA, Silver Spring, MD 1

Abstract

• Background: Clinical pharmacodynamic similarity data can contribute to the totality of evidence supporting the approval of biosimilars and can guide the need for subsequent clinical testing. Proteomic analysis of plasma samples from a clinical trial FDA conducted identified interferon-gamma-inducible protein-10 (IP-10) as a candidate pharmacodynamic (PD) biomarker of IFNβ-1 biologics (interferon beta-1a [IFNβ-1a] and pegylated IFNβ-1a [pegIFNβ-1a]).

• Purpose: To evaluate a quantitative immuno-PCR assay for the technical replication of plasma IP-10 levels.

• Study Design: We used plasma samples from 36 healthy subjects from the placebo-controlled randomized clinical trial carried out with IFNβ-1a and pegIFNβ-1a.

• Methods: A commercial assay (ProQuantum, ThermoFisher Scientific) was used to measure IP-10 at 10-time points over 6 days in the IFNβ-1a group
  (n=12 [30µg]), at 12 timepoints over 13 days in the pegIFNβ-1a group (n=12 [125µg]) and placebo-specific groups (n=6 each). Concentrations were extrapolated from a standard curve analysis. For differential expression, we conducted ANOVA on linear-mixed effect models. F-test and area under effect curve (AUEC). Results were compared to previous proteomics findings
  (SOMAscan Assay v4.1, SomaLogic) using Pearson’s Correlation. Analyses were conducted in R (version 4.1.2).

• Results: Median concentration of IP-10 in baseline plasma samples was 20.8 pg/ml. Assay sensitivity was 0.064 pg/ml, with 4 log-fold dynamic range and good reproducibility (CV<25%). F-test p-values of 2.9E-28 and 4.9E-30 for IFNβ1-a and pegIFNβ-1a products, respectively, were observed. At 0.33 days, IP-10 showed maximum response with a 38-fold and 28-fold increase from baseline for IFNβ1-a and pegIFNβ-1a, respectively. A strong difference compared to placebo was observed for AUECs for IFNβ-1a (t-test p=1.04E-04) and pegIFNβ-1a (p=8.6E-04). The pattern of IP-10 response for both products over time was concordant with previous proteomic analysis of the same plasma samples (rho=0.87, p=3.9E-122).

• Conclusion: Results suggest that the IP-10 assay is a sensitive and reproducible method and further technically replicates previous findings supporting IP-10 as a potential pharmacodynamic biomarker for IFNβ1-a products in healthy subjects. This assay has potential utility in future longitudinal studies for the detection of IP-10 in plasma as a candidate PD biomarker for assessing biosimilarity.

Introduction

• Pharmacodynamic (PD) biomarkers may be used to support biosimilar clinical development programs, without the need for comparative clinical studies with efficacy endpoints1,2.
• Interferon-beta-1a (INFβ-1a) and pegylated-IFNβ-1a (pegIFNβ-1a) are approved biologics drugs to treat multiple sclerosis and have complex pharmacology and limited PD biomarkers.
• Using plasma proteomics, we recently identified interferon-gamma-inducible protein-10 (IP-10) as a potential plasma PD biomarker of INFβ-1a and pegINFβ-1a.
• In the present study, our objective was to evaluate a quantitative immuno-polymerase chain reaction (iPCR) method for technical replication of plasma IP-10 response to both drugs in plasma over time to access the potential of IP-10 to be used as PD biomarker for the above-mentioned drugs.

Materials and Methods

Plasma samples were collected from a single-dose, randomized, placebo- controlled clinical study conducted by the FDA (U.S. Food and Drug Administration). Plasma IP-10 levels were measured using a Human IP-10 ProQuantum Immunoassay (Thermofisher Scientific)4 according to the manufacturer’s instructions. Concentrations were extrapolated from a standard curve analysis. For differential expression, we conducted ANOVA on linear- mixed effect models. The F-test p-value <0.05 for drug-time interaction was considered significant. Student’s t-test was conducted between AUEC (area under effect curve) of drug and placebo. Comparison between Proquantum assay results and previous proteomics findings (SOMAscan® Assay v4.1, SomaLogic)5 was conducted using Pearson’s correlation. Analyses were conducted in R (version 4.1.2).

Randomized, single-dose clinical study (NCT04183491)3 36 healthy subject

Disclaimer: The findings and conclusions in this presentation reflect the views of the authors and should not be construed to represent FDA’s views or policies. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.

Scan to access the full text of the article

Results and Discussion

• IP-10 was abundant (median=20.8 pg/ml) in baseline plasma samples.
• Assay sensitivity was 0.064 pg/ml, with 4 log-fold dynamic range and good reproducibility (CV<25%).
• IFNβ1-a and pegIFNβ-1a products, significantly impacted IP10 levels in plasma over time (Lmer, F-test p-values of 2.9E-28 and 4.9E-30, respectively).
• Max response in IP-10 levels was observed at 0.33 days with a 38-fold and 28-fold increase in levels from baseline for IFNβ1-a and pegIFNβ-1a, respectively. AUEC were significantly different for IP-10 for IFNβ-1a (t-test p=1.04E-04) and pegIFNβ-1a (p=8.6E-04) compared to placebo.
• Strong correlation between the two methods was observed (Pearson correlation, rho=0.87, p=3.9E-122).
• This assay has potential utility in future longitudinal detection of IP-10 in plasma as a candidate PD biomarker for biosimilarity for IFNβ1-a products.

ProQuantum IP-10 Response Curves

Plasma levels of IP-10 were significantly impacted by both INFß-1a biologics over the study time

Acknowledgements

Division of Applied Regulatory Science Omics Team, Office of Clinical Pharmacology Management, Biosimilar Guidance & PD biomarker working groups.
This project was supported, in part, by an appointment to the ORISE Research Participation Program at the CDER administered by the ORISE through an interagency agreement between the U.S. DoE and the U.S. FDA.

Proteomic IP-10 Response was Replicated using ProQuantum

Strong correlation between the two methods or technologies was also observed :
Pearson’s r value = 0.87
p-value = 3.93E-122
Figure 3. ProQuantum IP-10 response pattern replicates previous proteomic result.

Conclusion

• The IP-10 quantitative iPCR assay is a sensitive and reproducible high-throughput method for the detection of IP-10 in plasma.
• Results technically replicate previous proteomic findings supporting IP-10 as a potential PD biomarker for IFNβ1-a products.
• This assay has potential utility in future longitudinal detection of IP-10 in plasma as a candidate PD biomarker for biosimilarity for IFNβ1-a products.

References

1. Li J, Florian J, Campbell E, Schrieber SJ, Bai JPF, Weaver JL, et al. Advancing Biosimilar development using pharmacodynamic biomarkers in clinical pharmacology studies. Clin Pharmacol Ther. 2020;107(1):40-2.
2. US Food and Drug Administration. FDA Guidance: Clinical pharmacology data to support a demonstration of biosimilarity to a reference product. 2016. Accessed 1 May 2022.
3. https://clinicaltrials.gov/ct2/show/NCT04183491?term=IFNB+1a&draw=2&rank=7
4. Bourdon, David M., et al. The ProQuantum immunoassay platform offers high performance in a simplified workflow for low-level measurement of cytokines in human and mouse sera/plasma. (2018): 174-39.
5. https://mohanlab.bme.uh.edu/wp-content/uploads/2017/02/SSM-002-Rev-4-SOMAscan-Technical-White-Paper.pdf

References

• Pharmacodynamic Biomarkers to Support Biosimilar Development: Interferon Beta-1A Products - Full Text View - ClinicalTrials.gov

Read User Manual Online (PDF format)

Read User Manual Online (PDF format)  >>

Download This Manual (PDF format)

Download this manual  >>