Fda Quantitative Immuno Pcr Detection Instruction Manual (+ PDF)

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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:

Collect plasma samples from the subjects or patients following
standard protocols.
Prepare the assay reagents according to the provided
instructions.
Thaw the plasma samples and ensure they are at room temperature
before beginning the assay.
Label the microplate wells according to the sample
identification.
Add the appropriate volume of each plasma sample to the
designated wells.
Add the assay reagents to each well, following the specified
volumes and mixing instructions.
Seal the microplate and gently shake to ensure proper mixing of
the samples and reagents.
Incubate the microplate at the recommended temperature and
duration.
After incubation, wash the microplate wells thoroughly to
remove any unbound substances.
Add the detection reagents to each well, following the
specified volumes and mixing instructions.
Seal the microplate and gently shake to ensure proper mixing of
the detection reagents.
Incubate the microplate again at the recommended temperature
and duration.
After incubation, wash the microplate wells thoroughly to
remove any unbound detection reagents.
Add the substrate solution to each well, following the
specified volumes and mixing instructions.
Incubate the microplate for the final time at the recommended
temperature and duration.
Stop the reaction by adding the stop solution to each
well.
Measure the absorbance or fluorescence of each well using a
compatible plate reader.
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.

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Quantitative Immuno-PCR Detection of Plasma IP-10 Levels Over Time: Candidate Pharmacodynamic Biomarker of INF-1 Biologics

Deepti P. Samarth1, Lakshmi Manasa S. Chekka1, Barry Rosenzweig1, Jeffry Florian1, David G. Strauss1, Paula L. Hyland1
Division of Applied Regulatory Sciences, Office of Clinical Pharmacology (OCP) / Office of Translational Sciences (OTS) / Center for 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 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 IFN-1 biologics (interferon beta-1a [IFN-1a] and pegylated IFN-1a [pegIFN-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 IFN-1a and pegIFN-1a.
Methods: A commercial assay (ProQuantum, ThermoFisher Scientific) was used to measure IP-10 at 10 timepoints over 6 days in the IFN-1a group (n=12 [30µg]), at 12 timepoints over 13 days in the pegIFN-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 IFN1-a and pegIFN-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 IFN1-a and pegIFN-1a, respectively. A strong difference compared to placebo was observed for AUECs for IFN-1a (t-test p=1.04E-04) and pegIFN-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 IFN1-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 (INF-1a) and pegylated-IFN-1a (pegIFN-1a) are approved biologics drugs to treat multiple sclerosis and have complex pharmacology and limited PD biomarkers.
· Using plasma proteomics, we recently identified interferon-gammainducible protein-10 (IP-10) as a potential plasma PD biomarker of INF1a and pegINF-1a.
· In the present study, our objective was to evaluate a quantitative immunopolymerase 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, placebocontrolled 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 manufacturer’s instructions. Concentrations were extrapolated from a standard curve analysis. For differential expression, we conducted ANOVA on linear-mixed effect models. An 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).

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%). · IFN1-a and pegIFN-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 IFN1-a and pegIFN-1a, respectively. AUEC were significantly different for IP-10 for IFN-1a (t-test p=1.04E-04) and pegIFN-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 IFN1-a products.

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

IFN-1a
[Clinically relevant dose 30 g]

pegIFN-1a
[Clinically relevant dose 125 g]

ProQuantum IP-10 Response Curves
Plasma levels of IP-10 were significantly impacted by both INF-1a biologics over the study time
IFN-1a (Drug*Time lmer p-value = 2.9E-28)

Proteomic IP-10 Response was Replicated using ProQuantum

Placebo n=6

n=12

Placebo n=6

n=12

10 timepoints over 6 days

Plasma samples
IP-10 quantitative immuno-PCR assay

12 timepoints over 13 days

Detecting IP-10 Levels in Plasma using Quantitative Immuno-PCR (iPCR)
ProQuantum Assay (ThermoFisher Scientific)

qPCR

· High throughput · Low sample volume input · Good dynamic range · High sensitivity and
reproducibility

Proximity ligation (PLA) technology

Figure 1. Experimental workflow

Scan to access the full text of
the article

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.

38-fold

38-fold increase in
IP-10 levels from baseline for IFN1a at 0.33 days

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.

pegIFN-1a (Drug*Time lmer p-value = 4.9E-30)

Conclusion
· The IP-10 quantitative iPCR assay is a sensitive and reproducible highthroughput method for the detection of IP-10 in plasma.
· Results technically replicate previous proteomic findings supporting IP-10 as a potential PD biomarker for IFN1-a products.
· This assay has potential utility in future longitudinal detection of IP-10 in plasma as a candidate PD biomarker for biosimilarity for IFN1-a products.

28-fold increase in IP-10 levels from baseline for IFN1a at 0.33 days
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Figure 2. IP-10 response curves generate from ProQuantum IP-10 assay results.
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.

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-002Rev-4-SOMAscan-Technical-White-Paper.pdf