Immunopurification and Mass Spectrometric Quantification of the Active Form of a Chimeric Therapeutic Antibody in Human Serum

Dubois, Mathieu; Fenaille, François; Clément, Gilles; Lechmann, Martin; Tabet, Jean‐Claude; Ezan, Éric; Bécher, François

doi:10.1021/ac7021234

Cited by 137 publications

(84 citation statements)

References 35 publications

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“…When no protein or peptide purification step is employed, LC/MS sensitivity is significantly compromised due to matrix interference from the peptide-rich digest [24]. For high-sensitivity LC/MS applications, immunopurification is the most effective way to improve assay sensitivity and robustness by isolating target protein from matrix [25,26]. Further immunopurification of surrogate peptide from the digest could be carried out using an anti-peptide Ab [27].…”

Section: Resultsmentioning

confidence: 99%

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

Roos¹,

Chen²,

Vesapogu³

et al. 2016

J Appl Bioanal

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Although enzyme-linked immunosorbent assays and electrochemiluminescence (ECL) immunoassays are the most widely used platform for ADA detection, they may be compromised by drug interference. We describe here an alternate, free of drug interference, immunocapture-LC/MS methodology for detecting anti-protein XYZ antibody in cynomolgus monkey plasma. In our approach, ADA-protein XYZ complexes are captured by a mouse monoclonal anti-drug antibody on streptavidin magnetic beads and separated from monkey plasma by a magnet. After elution, ADA are digested with trypsin and detected by LC/MS using a surrogate peptide common to monkey IgG subclasses 1-4. The immunocapture-LC/MS assay was applied to support a toxicology study and results were in close agreement with those from a modified ECL immunoassay. To our knowledge, this is the first application of using LC/MS for monkey ADA detection.

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Section: Resultsmentioning

confidence: 99%

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

Roos¹,

Chen²,

Vesapogu³

et al. 2016

J Appl Bioanal

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“…There were a few publications using tandem high-performance chromatography mass spectrometry (LC-MS/MS) methods to quantify mAb (10)(11)(12)(13)(14)(15). The method approach involves enzyme digestion to reduce mAb into small peptides to be within the mass range of the mass spectrometry.…”

Section: Discussionmentioning

confidence: 99%

Bioanalytical Approaches to Quantify “Total” and “Free” Therapeutic Antibodies and Their Targets: Technical Challenges and PK/PD Applications Over the Course of Drug Development

Lee

Kelley

King

et al. 2011

AAPS J

240

151

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Abstract. The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono-and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.

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“…Biotherapeutics or associated surrogate peptides as well as any important catabolites could be specifically quantified by MS. In fact, several researchers have published studies with this ultimate goal in mind (Dubois et al, 2007(Dubois et al, , 2008Heudi et al, 2008;Ezan et al, 2009;Liu et al, 2011b;Bronsema et al, 2012;Li et al, 2012). Historically, there have been several drawbacks associated with quantitative MS methods compared with LBA, including the following: 1) MS has been generally less sensitive, 2) large protein analytes have suffered from low throughput, and 3) quantitative MS methods for macromolecules are much more nascent compared with established antibody-based methodologies.…”

Section: Impact Of Protein Biotherapeutic Biotransformation On Bioanamentioning

confidence: 99%

Biotransformation and In Vivo Stability of Protein Biotherapeutics: Impact on Candidate Selection and Pharmacokinetic Profiling

Hall

2014

Drug Metab Dispos

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Historically, since the metabolism of administered peptide/protein drugs ("biotherapeutics") has been expected to undergo predictable pathways similar to endogenous proteins, comprehensive biotherapeutic metabolism studies have not been widely reported in the literature. However, since biotherapeutics have rapidly evolved into an impressive array of eclectic modalities, there has been a shift toward understanding the impact of metabolism on biotherapeutic development. For biotherapeutics containing non-native chemical linkers and other moieties besides natural amino acids, metabolism studies are critical as these moieties may impart undesired toxicology. For biotherapeutics that are composed solely of natural amino acids, where end-stage peptide and amino acid catabolites do not generally pose toxicity concerns, the understanding of biotherapeutic biotransformation, defined as in vivo modifications such as peripherally generated intermediate circulating catabolites prior to end-stage degradation or elimination, may impact in vivo stability and potency/clearance. As of yet, there are no harmonized methodologies for understanding biotherapeutic biotransformation and its impact on drug development, nor is there clear guidance from regulatory agencies on how and when these studies should be conducted. This review provides an update on biotherapeutic biotransformation studies and an overview of lessons learned, tools that have been developed, and suggestions of approaches to address issues. Biotherapeutic biotransformation studies, especially for certain modalities, should be implemented at an early stage of development to 1) understand the impact on potency/clearance, 2) select the most stable candidates or direct protein re-engineering efforts, and 3) select the best bioanalytical technique(s) for proper drug quantification and subsequent pharmacokinetic profiling and exposure/response assessment.

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Immunopurification and Mass Spectrometric Quantification of the Active Form of a Chimeric Therapeutic Antibody in Human Serum

Cited by 137 publications

References 35 publications

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

Bioanalytical Approaches to Quantify “Total” and “Free” Therapeutic Antibodies and Their Targets: Technical Challenges and PK/PD Applications Over the Course of Drug Development

Biotransformation and In Vivo Stability of Protein Biotherapeutics: Impact on Candidate Selection and Pharmacokinetic Profiling

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