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Ligand Binding Assays in the 21st Century Laboratory: Recommendations for Characterization and Supply of Critical Reagents

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  • Theme: Ligand Binding Assays in the 21st Century Laboratory
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Abstract

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

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Abbreviations

Ab(s):

Antibody(ies)

ADA:

Anti-drug antibody

BA:

Bioanalytical

BSA:

Bovine serum albumin

CRO:

Contract research organization

DLS:

Dynamic light scattering

ECL:

Electro-chemiluminescence

GLP:

Good laboratory practices (21CFR part 58)

HCP:

Host cell protein

HPLC:

High-performance liquid chromatography

HRP:

Horseradish peroxidase

IgG:

Immunoglobulin G

IND:

Investigational new drug

KLH:

Keyhole limpet hemocyanin

LBA(s):

Ligand binding assay(s)

LC:

Liquid chromatography

LIMS:

Laboratory information management system

MAb(s):

Monoclonal antibody(ies)

MS:

Mass spectrometry

NAb(s):

Neutralizing antibody(ies)

PAb(s):

Polyclonal antibody(ies)

PC:

Positive control

PK:

Pharmacokinetics

QC(s):

Quality control(s)

S/B:

Signal-to-background ratio

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEC:

Size exclusion chromatography

SOP:

Standard operating procedure

SPR:

Surface plasmon resonance

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ACKNOWLEDGMENTS

We extend our thanks to all the reviewers of this manuscript, in particular, Jean Lee, Valerie Quarmby, Marian Kelley, all departmental reviewers, and the Ligand Binding Assay Bioanalytical Focus Group (LBABFG) of the American Association of Pharmaceutical Scientists (AAPS) steering committee for their critical review of this manuscript and helpful comments. We also thank Terri Caiazzo (Pfizer), Rosemary Lawrence-Henderson (Pfizer), Brian J. Geist (Janssen R&D, LLC), Michele Frigo (Janssen R&D, LLC), Tong-Yuan Yang (Janssen R&D, LLC), and Yanhong Li (Genentech/Roche) for providing data shown in the Electronic supplementary material S1. We acknowledge the AAPS Twenty-First Century Bioanalytical Laboratory Workshop: planning committee members Jean Lee (co-chair), Valerie Quarmby (co-chair), Ago Ahene, Marian Kelley, Sheldon Leung, Chad Ray, Huifen Faye Wang, Melvin Weinswig; the programming committee (Jean Lee and Valerie Quarmby); and the steering committee (Ago Ahene, Chad Ray and Sheldon Leung). This manuscript was prepared by members of the Twenty-First Century Bioanalytical Laboratory: Reagent Subcommittee of the LBABFG of AAPS.

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Authors and Affiliations

  1. Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc (formerly Wyeth), One Burtt Road, Andover, Massachusetts 01810, USA

    Denise M. O’Hara

  2. Clinical Assay Development, Genzyme, A Sanofi Company, 1 Mountain Rd, Framingham, Massachusetts, 01701, USA

    Valerie Theobald

  3. Department of Biologics Clinical Pharmacology, Janssen Research and Development, LLC, 145 King of Prussia Rd., Radnor, Pennsylvania 19087, USA

    Adrienne Clements Egan

  4. Product Management Department, Thermo Fisher Scientific, 1601 Cherry St., Philadelphia, Pennsylvania, 19102, USA

    Joel Usansky

  5. ABD-BioAnalytical Sciences, Biologics, Bristol-Myers Squibb Company, Route 206 and Provinceline Road, Princeton, New Jersey, 08543-4000, USA

    Murli Krishna

  6. Quality Control, Amgen, Inc, 4000 Nelson Road, Longmont, Colorado, 80503, USA

    Julie TerWee

  7. BioAnalytical Sciences Department, Genentech/Roche, 1 DNA Way, South San Francisco, California, 94080, USA

    Mauricio Maia

  8. Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer, Inc, One Burtt Road, Andover, Massachusetts 01810, USA

    Frank P. Spriggs

  9. Antibody Solutions, 1130 Mountain View Alviso Road, Sunnyvale, California, 94089-2221, USA

    John Kenney

  10. BioAnalytical Operations, BioAgilytix Labs, 2300 Englert Drive, Suites G-J, Research Triangle Park, Durham, North Carolina, 27713, USA

    Afshin Safavi

  11. Preclinical and Clinical Development Sciences, Biogen Idec Inc, 15 Cambridge Center, Cambridge, Massachusetts, 02142, USA

    Jeannine Keefe

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  1. Denise M. O’Hara
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  2. Valerie Theobald
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  3. Adrienne Clements Egan
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  4. Joel Usansky
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  5. Murli Krishna
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  6. Julie TerWee
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  7. Mauricio Maia
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  8. Frank P. Spriggs
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  9. John Kenney
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Corresponding author

Correspondence to Denise M. O’Hara.

Additional information

Guest Editors: William Nowatzke, Ago Ahene, and Chad Ray

Electronic supplementary material

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Fig. S1

Examples of reagent characterizations and assay performance. a A large quantity of a critical reagent was requested from a vendor who had previously supplied it using a soluble expression system in CHO cells. To supply material for the large request, the vendor changed the expression system to Escherichia coli, harvested inclusion bodies, and refolded the protein reagent. Performance of the E. coli expressed protein in an established LBA showed similar maximum and minimum signal responses compared with the earlier preparation. However, three times the amount of this protein was required to coat the plate and the IC50 of the ELISA changed by twofold relative to the LBA, using the reagent from the mammalian secreted process. Characterization of the reagents, derived from the different expression systems, by SEC showed that the reagent from the E. coli process eluted earlier and had an apparent higher molecular weight than the corresponding reagent from the CHO-derived process. Expressing, harvesting, and refolding protein reagents from bacterial inclusion bodies may have resulted in misfolded and/or aggregated material that was not present in the reagent lot from the soluble mammalian expression system. Qualification of the new reagent lot relative to the soluble mammalian expressed reagent demonstrated differences in performance of the LBA, and the SEC profile of the reagent provided a means to screen new lots of this critical reagent. b The biophysical state of an LBA capture reagent can affect assay performance. The example here is of a recombinant protein that naturally exists in a dimeric state. The protein was purified as monomeric and dimeric proteins and subsequently conjugated with biotin. Under identical conditions (10% mouse plasma) and concentrations, these capture reagents were coated onto streptavidin plates to compare their ability to capture a biotherapeutic recognizing the target protein. While the assay background remained the same, the signal/noise ratio was higher, by at least tenfold with the monomeric capture reagent than the dimeric capture reagent. c Asterisk: sheep polyclonal anti-human IgG antibody spiked into cynomolgus pooled serum. A bridging assay format was used, in which assay signal derived from ADA molecules that bridged two drug molecules, one drug molecule labeled with ruthenium, the other with biotin. ECL units were measured using a commercial chemiluminescence analyzer. A therapeutic MAb was conjugated with a ruthenium-label at different molar challenge ratios. The effect of different molar challenge ratios is evident in the LBA signal, which corresponds to electrochemiluminescence (ECL) units measured in an immunogenicity assay. The ADA specific for the therapeutic MAb was detected using a bridging assay format where the assay signal is obtained from molecules that bridged two labeled drug molecules (one labeled with ruthenium, the other labeled with biotin). The assay background and the positive-sample assay signal increased with increasing challenge ratios. The signal-to-background (S/B) ratio also increased with higher molar-ratio challenges, indicating the potential for improved method sensitivity with greater ratios of ruthenium to protein. d To develop a NAb assay, a positive control (PC) with neutralizing activity is required as a critical reagent. Several animals may be initially immunized initially to generate a PAb PC, and it is essential to gather information on the sample time points (bleeds) for neutralizing activity. In this assay, a dramatic decrease in IC50 in the LBA is observed as the neutralizing activity of PAbs matures with time a dramatic decrease in IC50 in the LBA is observed. This demonstrates that bleeds collected at different times from the same animal are not always equivalent in reactivity. It is important to maximize the amount of each lot of PAb reagent, perhaps by pooling several bleeds with appropriate activity. As part of a strategic plan to maintain the assay and to assure appropriate controls, subsequent lots may require a partial validation to determine new assay acceptance criteria for that new assay reagent. e Four lots of reagent Abs were produced and purified on separate occasions from the same hybridoma cell bank. The purification procedures for all lots were similar, and no differences between lots were observed when the Abs were evaluated by SEC or by reducing and non-reducing SDS-PAGE. The purified reagent Abs were then conjugated with ruthenium for use as a detection Ab in a PK assay. A conjugated MAb from Lot #301 was initially validated at a working concentration of 1.25 μg/mL in the PK assay, and attempts were made to qualify the subsequent lots at the same concentration. A conjugated antibody made from Lot #302 was qualified in the assay at an identical concentration as Lot #301. However, a conjugate made from Lot #303 could not be qualified in the assay and a conjugate made from Lot #304 could only be qualified in the assay at twice the concentration (2.5 μg/mL) of earlier lots. The source lots of these conjugated reagent antibodies were then evaluated for intact and reduced forms by LC-MS (reversed-phase HPLC coupled to time-of-flight mass spectrometry). The LC-MS experiments revealed that Lot # 301 and 302 had nearly identical spectra for their heavy chains, while a set of larger (∼1 kD shift) peaks were in the spectra from the heavy chains of Lots 303 and 304. These experiments indicate that as the relative signal of this higher mass species (when compared with the signal of the original mass) increased, the assay performance also decreased. Therefore, the LC-MS method could be used to prescreen subsequent lots for use in the assay (JPEG 70 kb)

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O’Hara, D.M., Theobald, V., Egan, A.C. et al. Ligand Binding Assays in the 21st Century Laboratory: Recommendations for Characterization and Supply of Critical Reagents. AAPS J 14, 316–328 (2012). https://doi.org/10.1208/s12248-012-9334-9

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