Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.

CAR-T therapies have shown remarkable efficacy against hematological malignancies in the clinic over the last decade and new studies indicate that progress is being made to use these novel therapies to target solid tumors as well as treat autoimmune disease. Innovation in the field, including TCR-T, allogeneic or "off the shelf" CAR-T, and autoantigen/armored CAR-Ts are likely to increase the efficacy and applications of these therapies. The unique aspects of these cell-based therapeutics; patient-derived cells, intracellular expression, in vivo expansion, and phenotypic changes provide unique bioanalytical challenges to develop pharmacokinetic and immunogenicity assessments. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Translational and ADME Sciences Leadership Group (TALG) has brought together a group of industry experts to discuss and consider these challenges. In this white paper, we present the IQ consortium perspective on the best practices and considerations for bioanalytical and immunogenicity aspects toward the optimal development of CAR-T and TCR-T cell therapies.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

The 2018 12(th) Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

Biological therapeutics are foreign antigens and can potentially induce immune response resulting in the formation of anti-drug antibodies (ADA), which in turn may lead to a wide range of side effects. Neutralizing Ab (NAb) is a subset of ADA that can bind to the pharmacological activity regions of therapeutic to inhibit or complete neutralize its clinical efficacy. A cell-based functional NAb assay is preferred to characterize its neutralization activity. However, cell-based NAb assays are often vulnerable to drug interference, as well as interference from numerous serum factors, including but not limited to growth factors and disease-related cytokines. Bead Extraction with Acid Dissociation (BEAD) has been successfully applied to remove circulating drug and/or other interfering factors from human serum samples, thereby enriching for ADA/NAb. However, the harsh acid used in the extraction procedure can cause irreversible denaturing of NAb and lead to underestimated NAb measurement. Herein we describe a new approach when acid-dissociation is not optimal for a PEGylated domain antibody (Ab). We further demonstrate that heating at 62 °C can not only dissociate drug/ADA/NAb immune complex but also selectively and irreversibly denature domain Ab drug due to much lower thermal stability of the domain Ab, when compared to that of full antibodies. The irreversible denaturing of the drug favors the formation of an immune complex between ADA/NAb and the added biotinylated drug thus increasing the recovery of ADA/NAb from samples. We call this new procedure Bead Extraction with Heat Dissociation (BEHD), which can potentially be applied to other NAb assays that have poor compatibility with acid dissociation.

AIM:A ligand-binding assay (LBA) was used to measure exposure of PRM-151, the recombinant form of human pentraxin-2 (PTX-2), a complex pentamer with multiple binding partners. However, the assay showed a lack of dose-dependent exposure in select preclinical species and it could not differentiate the infused PRM-151 from the endogenous PTX-2 in nonhuman primates.MATERIALS & METHODS:Instead of assessing interference from its multiple binding partners, which could be time consuming and laborious, a LC-MS assay avoid of these interference was implemented to measure 'total' drug without the use of immunoaffinity capture reagents.RESULTS & CONCLUSION:The resultant LC-MS data confirmed the original data and the lack of dose-dependent exposure is now understood to be due to the multiple and diverse targets and functions and resultant complex biodistribution rather than an assay artifact.

Ipilimumab is the first US FDA-approved immune checkpoint-blocking antibody drug to harness the patient's own immune cells. One of the postmarketing requirements is to develop a cell-based neutralizing antibody assay. Here, we share some of the most challenging aspects encountered during the assay development: new cell line construction; an unexpected inhibition of T-cell activation by low concentrations of ipilimumab; and two issues caused by sample pretreatment with acid dissociation to overcome drug interference: instability of neutralizing antibody positive control at low pH, and incompatibility of commonly used acid dissociation buffers in the cell assay. After troubleshooting and optimization, we successfully validated the assay and used the assay to test clinical samples to date.

Novel treatment options are needed to improve long-term outcomes for patients with multiple myeloma (MM). In this article, we comprehensively review the clinical pharmacology of elotuzumab, a first-in-class monoclonal anti-SLAMF7 antibody approved in combination with lenalidomide and dexamethasone (ELd) for the treatment of patients with MM and one to three prior therapies. Elotuzumab has a dual mechanism of action to specifically kill myeloma cells: binding SLAMF7 on myeloma cells facilitates natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), and direct engagement of SLAMF7 on NK cells further enhances NK cell activity. Elotuzumab administration causes transient elevations of selected cytokines (tumor necrosis factor-α, interferon-γ-induced protein-10 and monocyte chemoattractant protein-1). The temporary nature of these elevations (greatest after the first dose, with a trend to return to baseline by day 7) suggests a low likelihood of facilitating clinically meaningful drug–drug interactions. Elotuzumab exposure increases more than proportionally to dose and >80% SLAMF7 receptor occupancy is achieved with the approved elotuzumab 10 mg/kg regimen. Population pharmacokinetic data from 375 patients demonstrated weight-based dosing is appropriate for elotuzumab, and that ethnicity and hepatic/renal function have minimal effects on exposure. Exposure–response analysis of patients treated with ELd demonstrated that increased elotuzumab exposure does not elevate the risk of grade 3+ adverse events (AEs) or AEs leading to discontinuation/death. Elotuzumab antidrug antibodies occurred in 18.5% of patients treated with ELd or elotuzumab plus bortezomib and dexamethasone, but were generally transient and did not affect elotuzumab efficacy or safety. A monotherapy study indicated elotuzumab does not have clinically relevant effects on QT intervals.

The 2018 12th Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LC-MS, hybrid ligand binding assay (LBA)/LC-MS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC-MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.

Elotuzumab is a humanized, immunostimulatory anti-signaling lymphocytic activation molecule F7 (SLAMF7) IgG1 monoclonal antibody indicated in combination with lenalidomide and dexamethasone for patients with multiple myeloma (MM) who have received 1–3 prior therapies. We assessed the immunogenicity of elotuzumab as a monotherapy and in combination with bortezomib/dexamethasone and lenalidomide/dexamethasone in patients with MM in five clinical studies, including the pivotal ELOQUENT-2 trial (NCT01239797). Anti-drug antibody (ADA) prevalence was determined using a validated bridging assay. The prevalence of neutralizing antibodies (NAbs) was assessed in ADA-positive samples from ELOQUENT-2. Data from four trials of elotuzumab combined with lenalidomide/dexamethasone or bortezomib/dexamethasone (n = 390 evaluable patients) demonstrated that nine (2.3%) patients were ADA positive in baseline assays, 72 (18.5%) were ADA positive on-treatment or during follow-up, and two (0.5%) developed persistent ADAs. Patients treated with elotuzumab monotherapy had a higher incidence of elotuzumab ADAs than those on the combination therapy. In general, ADAs developed early and resolved after 2–4 months. Of 45 on-treatment ADA-positive patients in ELOQUENT-2, 19 had NAbs. Population pharmacokinetic modeling demonstrated an apparent increase in target-mediated elimination (higher Vmax, lower KM) in ADA-positive versus ADA-negative patients. ADAs were associated with lower elotuzumab steady-state exposure; however, this result may have been confounded by differential myeloma protein levels. ADAs/NAbs were not associated with hypersensitivity, infusion reactions, or loss of elotuzumab efficacy. Using a novel visualization, we also demonstrate that there is no clear relationship between the occurrence and titer values of ADA/NAbs and progression-free survival and best overall response status in patients treated with elotuzumab.

BioanalysisVol. 9, No. 21 CommentaryThe future of immunocapture-capillary electrophoresis-high resolution mass spectrometryLinna Wang, Robert W Dodge & Qin C JiLinna Wang Bioanalytical Sciences, Bristol-Myers Squibb, Rt. 206 & Province line Road, Princeton, NJ 08540, USA, Robert W Dodge Bioanalytical Sciences, Bristol-Myers Squibb, Rt. 206 & Province line Road, Princeton, NJ 08540, USA & Qin C Ji*Author for correspondence: Tel.: +1 609 252 5560; E-mail Address: qin.ji@bms.com Bioanalytical Sciences, Bristol-Myers Squibb, Rt. 206 & Province line Road, Princeton, NJ 08540, USAPublished Online:2 Nov 2017https://doi.org/10.4155/bio-2017-4977AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: capillary electrophoresisglycoproteinshigh resolution mass spectrometryimmunocaptureintact protein analysisLC–MSprotein biotransformation productsprotein drug variantsReferences1 Mahajan PB. Recent advances in application of pharmacogenomics for biotherapeutics. AAPS J. 18(3), 605–611 (2016).Crossref, Medline, CAS, Google Scholar2 Group BDW. Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework. Clin. Pharm. Ther. 69(3), 89–95 (2001).Crossref, Medline, Google Scholar3 Gucinski AC, Boyne MT 2nd. Evaluation of intact mass spectrometry for the quantitative analysis of protein therapeutics. Anal. Chem. 84(18), 8045–8051 (2012).Crossref, Medline, CAS, Google Scholar4 Ruan Q, Ji QC, Arnold ME, Humphreys WG, Zhu M. Strategy and its implications of protein bioanalysis utilizing high-resolution mass spectrometric detection of intact protein. Anal. Chem. 83(23), 8937–8944 (2011).Crossref, Medline, CAS, Google Scholar5 Hall MP, Gegg C, Walker K et al. Ligand-binding mass spectrometry to study biotransformation of fusion protein drugs and guide immunoassay development: strategic approach and application to peptibodies targeting the thrombopoietin receptor. AAPS J. 12(4), 576–585 (2010).Crossref, Medline, CAS, Google Scholar6 He L, Anderson LC, Barnidge DR, Murray DL, Hendrickson CL, Marshall AG. Analysis of monoclonal antibodies in human serum as a model for clinical monoclonal gammopathy by use of 21 Tesla FT-ICR top-down and middle-down MS/MS. J. Am. Soc. Mass Spectrom. 28(5), 827–838 (2017).Crossref, Medline, CAS, Google Scholar7 Jacobs JF, Wevers RA, Lefeber DJ, Van Scherpenzeel M. Fast, robust and high-resolution glycosylation profiling of intact monoclonal IgG antibodies using nanoLC-chip-QTOF. Clin. Chim. Acta 461, 90–97 (2016).Crossref, Medline, CAS, Google Scholar8 Schachner L, Han G, Dillon M et al. Characterization of chain pairing variants of bispecific IgG expressed in a single host cell by high-resolution native and denaturing mass spectrometry. Anal. Chem. 88(24), 12122–12127 (2016).Crossref, Medline, CAS, Google Scholar9 Van Den Broek I, Van Dongen WD. LC–MS-based quantification of intact proteins: perspective for clinical and bioanalytical applications. Bioanalysis 7(15), 1943–1958 (2015).Link, CAS, Google Scholar10 Petersen JR, Mohammad AA. Clinical And Forensic Applications Of Capillary Electrophoresis. Humana Press, NJ, USA (2001).Crossref, Google Scholar11 Faserl K, Sarg B, Maurer V, Lindner HH. Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry. J. Chromatogr. A 1498, 215–223 (2017).Crossref, Medline, CAS, Google Scholar12 Khatri K, Klein JA, Haserick JR et al. Microfluidic capillary electrophoresis–mass spectrometry for analysis of monosaccharides, oligosaccharides, and glycopeptides. Anal. Chem. 89(12), 6645–6655 (2017).Crossref, Medline, CAS, Google Scholar13 Zhang Z, Albanetti T, Linkous T et al. Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry. Electrophoresis 38(3–4), 401–407 (2017).Crossref, Medline, CAS, Google Scholar14 Lloyd DK. Capillary electrophoresis analysis of biofluids with a focus on less commonly analyzed matrices. J. Chromatogr. B 866(1–2), 154–166 (2008).Crossref, Medline, CAS, Google Scholar15 Ramautar R, Somsen GW, De Jong GJ. Direct sample injection for capillary electrophoretic determination of organic acids in cerebrospinal fluid. Anal. Bioanal. Chem. 387(1), 293–301 (2007).Crossref, Medline, CAS, Google Scholar16 Naylor S, Ji Q, Johnson KL, Tomlinson AJ, Kieper WC, Jameson SC. Enhanced sensitivity for sequence determination of major histocompatibility complex class I peptides by membrane preconcentration–capillary electrophoresis–microspray–tandem mass spectrometry. Electrophoresis 19(12), 2207–2212 (1998).Crossref, Medline, CAS, Google Scholar17 Kartsova LA, Bessonova EA. Preconcentration techniques in capillary electrophoresis. J. Anal. Chem. 64(4), 326–337 (2009).Crossref, CAS, Google Scholar18 Zhang H, Xiao Q, Xin B et al. Development of a highly sensitive liquid chromatography/tandem mass spectrometry method to quantify total and free levels of a target protein, interferon-gamma-inducible protein-10, at picomolar levels in human serum. Rapid Commun. Mass Spectrom. 28(13), 1535–1543 (2014).Crossref, Medline, CAS, Google Scholar19 CytomX Therapeutics. http://Cytomx.Com/Probody-Therapeutics/#Av_Section_3.Google Scholar20 Reis CA, Osorio H, Silva L, Gomes C, David L. Alterations in glycosylation as biomarkers for cancer detection. J. Clin. Pathol. 63(4), 322–329 (2010).Crossref, Medline, CAS, Google Scholar21 Locker GY, Hamilton S, Harris J et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J. Clin. Oncol. 24(33), 5313–5327 (2006).Crossref, Medline, CAS, Google Scholar22 Ebeling FG, Stieber P, Untch M et al. Serum CEA and CA 15–3 as prognostic factors in primary breast cancer. Br. J. Cancer 86(8), 1217–1222 (2002).Crossref, Medline, CAS, Google Scholar23 Kumpulainen EJ, Keskikuru RJ, Johansson RT. Serum tumor marker CA 15.3 and stage are the two most powerful predictors of survival in primary breast cancer. Breast Cancer Res. Treat. 76(2), 95–102 (2002).Crossref, Medline, CAS, Google Scholar24 Pinho SS, Reis CA. Glycosylation in cancer: mechanisms and clinical implications. Nat. Rev. Cancer 15(9), 540–555 (2015).Crossref, Medline, CAS, Google Scholar25 Sato Y, Nakata K, Kato Y et al. Early recognition of hepatocellular carcinoma based on altered profiles of alpha-fetoprotein. N. Engl. J. Med. 328(25), 1802–1806 (1993).Crossref, Medline, CAS, Google Scholar26 Noda K, Miyoshi E, Uozumi N et al. Gene expression of alpha1–6 fucosyltransferase in human hepatoma tissues: a possible implication for increased fucosylation of alpha-fetoprotein. Hepatology 28(4), 944–952 (1998).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByApplications of capillary electromigration methods for separation and analysis of proteins (2017–mid 2021) – A reviewAnalytica Chimica Acta, Vol. 1209Themed issue on 'emerging technologies in mass spectrometry'J Larry Campbell & Robert Plumb17 November 2017 | Bioanalysis, Vol. 9, No. 21 Vol. 9, No. 21 Follow us on social media for the latest updates Metrics Downloaded 139 times History Received 22 August 2017 Accepted 7 September 2017 Published online 2 November 2017 Published in print November 2017 Information© 2017 Future Science LtdKeywordscapillary electrophoresisglycoproteinshigh resolution mass spectrometryimmunocaptureintact protein analysisLC–MSprotein biotransformation productsprotein drug variantsDisclaimerThe views and opinions expressed in this article are those of the authors and do not necessarily reflect the position of authors' employer.Financial & competing interest disclosureThe authors of this article are current employees of Bristol-Myers Squibb Company (BMS). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Gene and nucleic acid therapies have demonstrated patient benefits to address unmet medical needs. Beside considerations regarding the biological nature of the gene therapy, the quality of bioanalytical methods plays an important role in ensuring the success of these novel therapies. Inconsistent approaches among bioanalytical labs during preclinical and clinical phases have been observed. There are many underlying reasons for this inconsistency. Various platforms and reagents used in quantitative methods, lacking of detailed regulatory guidance on method validation and uncertainty of immunogenicity strategy in supporting gene therapy may all be influential. This review summarizes recent practices and considerations in bioanalytical support of pharmacokinetics/pharmacodynamics and immunogenicity evaluations in gene therapy development with insight into method design, development and validations.

PEGylation is a proven approach to prolonging the duration of action and enhancing biophysical solubility and stability of peptides. 4-Acetylphenylalanine is a novel amino acid with a ketone side chain that is uniquely reactive in proteins. The ketone functionality can react with an aminooxy functionalized polyethyleneglycol polymer to form a stable oxime adduct of the protein. One concern with using unnatural amino acids, such as 4-acetylphenylalanine, is the possibility of it being cleaved from the peptide and becoming incorporated into endogenous proteins. To determine whether this occurs, an in vitro experiment to assess the cell viability and amino acid incorporation into endogenous proteins using primary male rat hepatocytes in the presence of [14 C]4-acetylphenylalanine, 4 or [14 C(U)]L-phenylalanine was conducted. [14 C]4-acetylphenylalanine, 4 was prepared in 2 radiochemical steps from [1-14 C]acetyl chloride in an overall 8% radiochemical yield and in 99.9% radiochemical purity. The results showed that there was no evidence of carbon-14 incorporation into hepatocyte endogenous proteins with [14 C]pAcF and there was no difference between it and L-phenylalanine in cell viability assessments at any of the concentrations studied between 0.1 and 1000 μM.

BACKGROUND:Polyethylene glycol (PEG) is a polymer that can be conjugated with therapeutic proteins. Monitoring anti-PEG antibodies in human subjects may be required as part of immunogenicity assessment. The lack of well-characterized anti-PEG reagents have limited our understanding of anti-PEG humoral response.RESULTS:Antibodies reactive to PEG were engineered with a human IgG1 Fc. Surface plasmon resonance and plate-based methods demonstrated that their binding was dependent on molecular weight (MW) of PEG. Specificity experiments using chemical analogs identified their specificity.CONCLUSION:Affinity, specificity and MW of PEG are critical characteristics that impact interactions of anti-PEG antibodies with PEG. These attributes especially MW of PEG and the assay formats may impact the ability to detect anti-PEG antibodies.

BACKGROUND:Extensive use of polyethylene glycol (PEG) in consumer products necessitates the assessment of anti-PEG antibodies (APAb).METHODS:In clinical trials comparing PEG-IFN-λ to PEG-IFN-α, conventional bridge and direct assays were assessed.RESULTS & CONCLUSION:The bridge assay detected IgM and IgG APAb reactive with common PEG sizes and derivatives at sufficient sensitivity, 15-500 ng/ml. Of subjects evaluated, 6% of PEG-IFN-λ and 9% of PEG-IFN-α subjects had persistent APAb while 60% of PEG-IFN-λ and 33% of PEG-IFN-α subjects had persistent anti-interferon antibodies (AIAb). Pre-existing APAb and AIAb prevalence was comparable (approximately 10% of subjects). APAb were earlier onset, less frequent, less persistent and lower titer than AIAb. No associated hypersensitivity events were reported.

Immunogenicity testing for PEGylated biotherapeutics should include methods to detect both anti-protein and anti-PEG antibodies (anti-PEG). Although some methods have been published for the detection of anti-PEG antibodies, the information is incomplete and, in some cases, reagents used (such as Tween-20) are known to interfere with detection. This rapid communication describes the use of BioScale’s Acoustic Membrane MicroParticle (AMMP®) technology using the ViBE® Workstation to measure anti-PEG antibodies in human serum samples. Briefly, a sample spiked with monoclonal human IgG anti-PEG antibody is diluted in buffer and incubated with paramagnetic beads coated with linear chain mPEG to capture anti-PEG antibodies. The complex is then captured on an acoustic membrane coated with Protein A. The change in mass on the membrane caused by the binding of the complex to the membrane results in a signal proportional to the mass of anti-PEG antibodies. The data indicate that an assay with a sensitivity of less than 1000 ng/mL for IgG is achievable. This level of sensitivity is better than current published reports on IgG anti-PEG antibody detection.

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC–MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing.

Immunogenicity testing for antidrug antibodies (ADA) faces challenges when high levels of the drug are present in clinical patient samples. In addition, most functional cell-based assays designed to characterize the neutralizing ability of ADA are vulnerable to interference from endogenous serum components. Bead extraction and acid dissociation (BEAD) has been successfully applied to extract ADA from serum samples prior to conduction of cell-based assays. However, in the BEAD, certain amounts of the drug and endogenous serum components (so-called residual drug and serum components) from serum samples are carried over to final BEAD eluates due to formation of protein complexes with ADA or nonspecific binding with the beads. Using current enzyme-linked immunosorbent assay (ELISA)-based ligand-binding assays, it is difficult to evaluate the residual drug, which is complexed with excessive amounts of ADA and endogenous serum components in the BEAD eluates. Here, we describe an innovative application of LC-MS/MS for simultaneous detection of the residual human monoclonal antibody drug and endogenous human IgG and the neutralizing antibody positive-control (NAb-PC) in the BEAD eluates. In this study, the low levels of the residual drug and human IgG in the BEAD eluates indicate that the BEAD efficiently removed the high-concentration drug and serum components from the serum samples. Meanwhile, the NAb-PC recovery (∼42%) in the BEAD provided an acceptable detection limit for the cell-based assay. This novel application of LC-MS/MS to immunogenicity assay development demonstrates the advantages of LC-MS/MS in selectivity and multiplexing, which provides direct and fast measurements of multiple components for immunogenicity assay development.