Simultaneous isotyping and semi-quantitation of anti-drug antibodies to an IgG1 biotherapeutic using hybrid LBA-LC-MS/MS.

Background: Commonly, ligand-binding platforms are being used for immunogenicity assessment, but with the recent advent of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for protein quantification, this technology has become an alternative for the measurement of anti-drug antibodies (ADAs), when combined with an immunocapture step to extract them out of the biological sample.

Method: The monoclonal antibody adalimumab was immobilized on magnetic beads to isolate ADAs against this drug from serum samples. Multiple repetitions of immunopurification were used to minimize nonspecific binding and improve drug tolerance while maintaining sufficient recovery.

Perspective on LC-MS(/MS) for biotherapeutic and biomarker proteins in research and regulated Bioanalysis: a consolidation of more than a decade of experience across the European Bioanalysis Forum community (Part 1: "The What").

Following up on our most recent discussion paper focusing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e.

A validated LC-MS/MS method for the simultaneous quantification of iothalamate and hippuran in serum and urine for non-radioactive kidney function assessment.

A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the kidney function markers iothalamate and hippuran in human serum and urine. It is based on protein precipitation with methanol followed by dilution of the supernatant for serum and simple dilution for urine. The polar analytes are chromatographically separated by a 6.

Simultaneous quantification of the 22-kDa isoforms of human growth hormone 1 and 2 in human plasma by multiplexed immunocapture and LC-MS/MS.

An LC-MS/MS method is presented for the simultaneous quantification of two structurally closely related protein biomarker isoforms, the 22-kDa isoforms of human growth hormone 1 and human growth hormone 2, in human plasma. It is based on multiplexed immunocapture using two monoclonal antibodies immobilized on magnetic beads, tryptic digestion and quantification of two specific signature peptides plus an additional peptide for estimation of total growth hormone related concentrations. A full validation according to international guidelines was performed across the clinically relevant concentration ranges of 0.

Quantitative bioanalysis of proteins by digestion and LC-MS/MS: the use of multiple signature peptides.

The use of multiple signature peptides for the quantification of proteins by digestion and LC-MS/MS is reviewed and evaluated here. A distinction is made based on the purpose of the use of multiple peptides: confirmation of the protein concentration, discrimination between different protein forms or species and biotransformation. Most reports that describe methods with at least two peptides use these for confirmation, but it is not always mentioned how the peptides are used and how possible differences in concentration between the peptides are handled.

Immunocapture LC-MS(/MS) assays for biotherapeutic and biomarker proteins: the European Bioanalysis Forum continuing discussions on scientific and regulatory challenges.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e.

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding.

Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation.

Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ion-exchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C.

An antibody-free LC-MS/MS method for the quantification of sex hormone binding globulin in human serum and plasma.

Objectives: Sex hormone binding globulin (SHBG) is a hormone binding protein which plays an important role in regulating the transport and availability of biologically active androgens and estradiol to target cells and used to calculate free testosterone concentrations.

Methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, featuring an albumin removal step followed by a tryptic digestion. After a reduction step with dithiothreitol and alkylation with iodoacetamide three signature peptides were used for the quantification of SHBG.

A Quantitative LC-MS/MS Method for Distinguishing the Tau Protein Forms Phosphorylated and Nonphosphorylated at Serine-396.

Hyperphosphorylated tau protein is well-known to be involved in the formation of neurofibrillary tangles and the progression of age-related neurodegenerative diseases (tauopathies), including Alzheimer's Disease (AD). Tau protein phosphorylated at serine-396 (pS396-tau) is often linked to disease progression, and we therefore developed an analytical method to measure pS396-tau in cerebrospinal fluid (CSF) in humans and animal models of AD. In the S396-region, multiple phosphorylation sites are present, causing structural complexity and sensitivity challenges for conventional bottom-up mass spectrometry approaches.

Revealing charge heterogeneity of stressed trastuzumab at the subunit level.

Trastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 °C) increases charge heterogeneity further.

Biotransformation of Trastuzumab and Pertuzumab in Breast Cancer Patients Assessed by Affinity Enrichment and Ion-Exchange Chromatography.

Therapeutic proteins (TPs) are known to be heterogeneous due to modifications that occur during the production process and storage. Modifications may also occur in TPs after their administration to patients due to in vivo biotransformation. Ligand binding assays, which are widely used in the bioanalysis of TPs in body fluids, are typically unable to distinguish such modifications.

Selective quantification of the 22-kDa isoform of human growth hormone 1 in serum and plasma by immunocapture and LC-MS/MS.

The human growth hormone GH1 (22 kDa) is a commonly measured biomarker for diagnosis and during treatment of growth disorders, but its quantification by ligand binding assays may be compromised by the occurrence of a number of isoforms. These can interfere in the assays and lead to differences in results between laboratories and potentially even in the treatment of patients. We present an LC-MS/MS method that is able to distinguish the major growth hormone isoform (GH1, 22 kDa) from other isoforms and quantify it without any interference across the clinically relevant concentration range of 0.

Effect of Trastuzumab-HER2 Complex Formation on Stress-Induced Modifications in the CDRs of Trastuzumab.

Asparagine deamidation and aspartic acid isomerization in the complementarity determining regions (CDRs) of monoclonal antibodies may alter their affinity to the target antigen. Trastuzumab has two hot spots for deamidation and one position for isomerization in the CDRs. Little is known how complex formation with its target antigen HER2 affects these modifications.

Analytical and pharmacological consequences of the in vivo deamidation of trastuzumab and pertuzumab.

A liquid chromatography-tandem mass spectrometry method is presented for the quantitative determination of the in vivo deamidation of the biopharmaceutical proteins trastuzumab and pertuzumab at an asparagine in their complementarity determining regions (CDRs). For each analyte, two surrogate peptides are quantified after tryptic digestion of the entire plasma protein content: one from a stable part of the molecule, representing the total concentration, and one containing the deamidation-sensitive asparagine, corresponding to the remaining non-deamidated concentration. Using a plasma volume of 10 µL and a 2-h digestion at pH 7, concentrations between 2 and 1000 µg/mL can be determined for the various protein forms with values for bias and CV below 15% and without unacceptable in vitro deamidation taking place.

Non-Antibody-Based Binders for the Enrichment of Proteins for Analysis by Mass Spectrometry.

There is often a need to isolate proteins from body fluids, such as plasma or serum, prior to further analysis with (targeted) mass spectrometry. Although immunoglobulin or antibody-based binders have been successful in this regard, they possess certain disadvantages, which stimulated the development and validation of alternative, non-antibody-based binders. These binders are based on different protein scaffolds and are often selected and optimized using phage or other display technologies.

Recommendations and discussion points on immunogenicity, biomarkers, automation/technology and protein-MS from the 2021 European Bioanalysis Forum Focus Workshops.

During the first half of 2021, and due to the SARS-CoV-2 pandemic preventing in-person meetings, the European Bioanalysis Forum organized four workshops as live interactive online meetings. The themes discussed at the workshops were carefully selected to match the cyberspace dynamics of the meeting format. The first workshop was a training day on challenges related to immunogenicity.

Enrichment and Liquid Chromatography-Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents.

Trastuzumab and pertuzumab are monoclonal antibodies used in the treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer. Therapeutic proteins may undergo chemical modifications that may affect the results of bioanalytical assays, as well as their therapeutic efficacy. Modifications may arise during production and storage, as well as after administration to patients.

Intact protein quantification in biological samples by liquid chromatography - high-resolution mass spectrometry: somatropin in rat plasma.

The quantitative determination of intact proteins in biological samples by LC with high-resolution MS detection can be a useful alternative to ligand-binding assays or LC-MS-based quantification of a surrogate peptide after protein digestion. The 22-kDa biopharmaceutical protein somatropin (recombinant human growth hormone) was quantified down to 10 ng/mL (0.45 nM) in 75 μL of rat plasma by the combination of an immunocapture step using an anti-somatropin antibody and LC-MS on a quadrupole-time of flight instrument.

Very complex internal standard response variation in LC-MS/MS bioanalysis: root cause analysis and impact assessment.

Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC-MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency.