Comparing singlet and duplicate immunogenicity assay in human plasma for pembrolizumab using Gyrolab®

Aim: For decades, the traditional approach for ligand-binding assays has been to generate two measurements from adjacent wells on the plate. In recent years, scientists have investigated the true benefit of this ‘duplicate analysis’ by looking back at previously generated bioanalytical data with the conclusion that the benefits are negligible. Materials & methods: We demonstrated a method development approach to determine the best number of replicate measurements of an immunogenicity assay. We used an anti-pembrolizumab immunogenicity assay on Gyrolab® to challenge the traditional use of duplicate measurements as we compare it to singlet measurement and show a balanced design for assessing the cut-point in singlet. Results & conclusion: We introduced the concept of calculating the maximum drug tolerance during method development. In this method, we found no practical benefit for duplicate analysis and go further in recommending that singlet analysis should be considered the default for all ligand-binding assays.

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Workshop Report: AAPS Workshop on Method Development, Validation, and Troubleshooting of Ligand-Binding Assays in the Regulated Environment

The AAPS Journal ◽

10.1208/s12248-015-9767-z ◽

2015 ◽

Vol 17 (4) ◽

pp. 1019-1024 ◽

Cited By ~ 2

Author(s):

Marian Kelley ◽

Lauren Stevenson ◽

Michaela Golob ◽

Viswanath Devanarayan ◽

Joao Pedras-Vasconcelos ◽

...

Keyword(s):

Ligand Binding ◽

Method Development ◽

Binding Assays ◽

Workshop Report

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Adventures in critical reagent lot changes in ligand-binding assays: redevelopment, bridging and additional processing requirements

Bioanalysis ◽

10.4155/bio-2020-0216 ◽

2021 ◽

Author(s):

Alissa Rauwerdink ◽

Michael Benson ◽

Allison Jayne ◽

Sathyapriya Babu ◽

Jessica St Charles ◽

...

Keyword(s):

Ligand Binding ◽

Method Development ◽

Close Monitoring ◽

Binding Assays ◽

Entire Study ◽

First Case ◽

Assay Performance ◽

And Performance ◽

The Impact

Aim: Critical reagents have significant impact on ligand-binding assay performance. The critical reagents selected during method development should be well-evaluated, as the quality of these reagents will dictate performance of the assay over time. Critical reagents in ligand-binding assays are almost always produced using a biological system, so batch yield, purity and performance tend to vary greatly. Due to the essential nature of critical reagents in the assay, changes in critical reagents can have dramatic impact on the assay and results, so close monitoring of assay performance is required. Methodology & results: We present here three examples of critical reagent lot changes that required creative solutions to maintain assay performance. The first case study is an example of the impact of different lots of analyte within a quantitative assay that resulted in the need to redevelop the assay in a different format. Case study two outlines an assay where a surrogate matrix is the critical reagent in an assay and the difficulties encountered over the course of several years and lot changes. The third case study covers an immunogenicity assay with a commercial detection that did not have sufficient quantity to cover the entire study lifecycle. As a result of the reagent change, a new assay was developed. Discussion & conclusion: A robust plan for critical reagent generation and lifecycle management should be adapted in order to avoid costly delays and rework. The performance of an assay depends on the continuity of the critical reagent supply. Reagents should be carefully selected to include the binding and performance properties required for an assay.

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Critical reagents for ligand-binding assays: process development methodologies to enable high-quality reagents

Bioanalysis ◽

10.4155/bio-2021-0217 ◽

2022 ◽

Author(s):

Caroline Kittinger ◽

Jared Delmar ◽

Lisa Hewitt ◽

Rebecca Holcomb ◽

Christopher Jones ◽

...

Keyword(s):

Ligand Binding ◽

Process Development ◽

Process Conditions ◽

Generation Process ◽

Binding Assays ◽

Performance Control ◽

Development Approach ◽

Immunogenicity Assays ◽

The Impact

Development of biotherapeutics require pharmacokinetic/pharmacodynamic (PK/PD) and immunogenicity assays that are frequently in a ligand-binding assay (LBA) format. Conjugated critical reagents for LBAs are generated conjugation of the biotherapeutic drug or anti-drug molecule with a label. Since conjugated critical reagent quality impacts LBA performance, control of the generation process is essential. Our perspective is that process development methodologies should be integrated into critical reagent production to understand the impact of conjugation reactions, purification techniques and formulation conditions on the quality of the reagent. In this article, case studies highlight our approach to developing process conditions for different molecular classes of critical reagents including antibodies and a peptide. This development approach can be applied to the generation of future conjugated critical reagents.

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The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

Scientific Reports ◽

10.1038/s41598-020-78926-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kenichi Kamata ◽

Kenji Mizutani ◽

Katsuya Takahashi ◽

Roberta Marchetti ◽

Alba Silipo ◽

...

Keyword(s):

Immune System ◽

Sialic Acid ◽

Ligand Binding ◽

Steric Hindrance ◽

Sequence Similarity ◽

Binding Assays ◽

Subunit Interface ◽

Nmr Techniques ◽

And Control ◽

Asialo Gm1

AbstractSeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac$$\alpha$$ α (2-3)Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc) and its precursor, asialo-GM1 (Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the $$\beta$$ β -trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

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