Middle-up quantification of therapeutic monoclonal antibodies in human plasma with two dimensional liquid chromatography high resolution mass spectrometry: Adalimumab as a proof of principle

Next generation human therapeutic monoclonal antibodies (t-mAbs) are harder to quantify with the widely used bottom-up tryptic digestion method. Due to their homology with endogenous immunoglobulins, there is a lack of unique and stable 'signature' peptides that can be targeted. Middle-up two dimensional liquid chromatography high resolution mass spectrometry (2D-LC-HRMS), targeting the entire light chain, was examined as an alternative. Adalimumab (ADM) was successfully quantified in human plasma after Melon® Gel sample purification, followed by orthogonal separation on a weak cation exchange (WCX) and reversed phase column. Charge and hydrophobicity were used to separate ADM from the polyclonal immunoglobulin background. HRMS with its high resolution and exact mass was able to isotopically resolve the ADM light chain and to provide another separation dimension on the basis of mass to charge ratio. Using the targeted single ion monitoring (T-SIM) with multiplex (MSX) option, three ADM light chain precursors, 2341.80, 2129.00, and 1951.68 m/z, and one internal standard precursor 2146.39 m/z, were measured simultaneously. The Melon® Gel sample purification was found to be very efficient in removing plasma proteins that would otherwise interfere with chromatographic separation and ionization. The linearity of the method for the analysis of ADM was excellent (R2=0.999) between 1 - 128 mg/L with an LLOQ signal to noise ratio (S/N) of 10. Within-run and between-run precision and accuracy were in concordance with the EMA guideline. Cross-validation of the 2D-LC-HRM method with the standard peptide LC-MS/MS method showed a good agreement (R2 = 0.86) between the methods. However, there was a bias present, possibly due to charge variant ADM formation over time. Since the presented 2D-LC-HRMS method is able to measure only the native form of ADM, it is able to provide a measure of the active form of ADM in patients

A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry

Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 - 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum

Optimization of a Quantitative Anti-Drug Antibodies against Infliximab Assay with the Liquid Chromatography-Tandem Mass Spectrometry: A Method Validation Study and Future Perspectives

Monoclonal antibodies (mAbs), such as infliximab, are important treatment options for different diseases. Immunogenicity is a major risk, resulting in anti-drug antibodies (ADAs), being associated with adverse events and loss of response, influencing long-term outcomes. The development of ADAs against infliximab is primarily measured by immunoassays like radioimmunoassay (RIA). Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized across different fields, this technique is currently not used for ADAs against infliximab measurements. Therefore, we developed the first LC-MS/MS method. Stable isotopically labeled infliximab antigen-binding fragments (SIL IFX F(ab')2) were used to bind and measure ADAs indirectly. Protein A magnetic beads were used to capture IgG, including ADAs, whereafter SIL IFX F(ab')2 was added for labeling. After washing, internal standard addition, elution, denaturation and digestion samples were measured by LC-MS/MS. Internal validation showed good linearity between 0.1 and 16 mg/L (R2 > 0.998). Sixty samples were used for cross-validation with RIA, and no significant difference between ADA concentrations was found. The methods had high correlation (R = 0.94, p < 0.001) and excellent agreement, intraclass correlation coefficient = 0.912 (95% confidence interval 0.858-0.947, p < 0.001). We present the first ADA against the infliximab LC-MS/MS method. The method is amendable for quantifying other ADAs, making it applicable as a template for future ADA methods

Quantification of emicizumab by mass spectrometry in plasma of people with hemophilia A: A method validation study

Background: Emicizumab is a new treatment option for people with hemophilia A. Emicizumab was approved with a body-weight-based dosage regimen, without laboratory monitoring requirements. Guidelines, however, recommend measuring emicizumab concentrations when the presence of antidrug antibodies is suspected. Furthermore, drug monitoring can be useful in clinical decision making, in adherence checking, and for research purposes. Therefore, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantifying emicizumab. We performed a validation study on this LC-MS/MS method quantifying emicizumab in the plasma of people with hemophilia A. Methods: Sample preparation for LC-MS/MS analysis included ammonium sulfate protein precipitation and trypsin digestion. A signature peptide of emicizumab and a matching stable isotope-labeled internal standard were used to quantify emicizumab by LC-MS/MS analysis. Validation was performed in accordance with the “Guideline on Bioanalytical Method Validation” of the European Medicines Agency (EMA). The LC-MS/MS method was cross validated against a modified and calibrated (r2 Diagnostics) one-stage clotting assay (OSA). Conclusions: The LC-MS/MS method demonstrated linearity over a wide range of emicizumab concentrations, far exceeding the concentrations observed in people with hemophilia A. Precision and accuracy were excellent, and all other validation parameters were also within the acceptance EMA criteria. Cross validation showed that the LC-MS/MS method and the OSA-based method can be used interchangeably for drug monitoring of emicizumab without the application of a correction factor

Ocular surface disease in moderate-to-severe atopic dermatitis patients and the effect of biological therapy

Atopic dermatitis (AD) is a chronic inflammatory skin disease for which new targeted therapies are currently available. Due to the increased rates of ocular surface disease (OSD) reported during treatment with these new targeted treatments, more insight into the occurrence and pathomechanism of OSD in moderate-to-severe AD patients is needed. Therefore, this review's first part highlights that most patients with moderate-to-severe AD already have characteristics of OSD before starting targeted treatment. Remarkably, not all AD patients with OSD report ocular symptoms. OSD in AD is associated with less conjunctival goblet cells (GC) compared to healthy controls. In addition, OSD severity in AD patients is associated with high AD activity, the presence of eyelid and/or facial eczema, and high levels of AD-related severity biomarkers in tear fluid. The second part of this review highlights that pre-existing ocular pathology (e.g. in combination with the use of ophthalmic medication or eyelid eczema) may be associated with the development of dupilumab-associated ocular surface disease (DAOSD). During dupilumab treatment, DAOSD (which can be new-onset OSD or worsening of pre-existing OSD) is observed in approximately one-third of the dupilumab-treated AD patients. Anti-inflammatory ophthalmic treatment improves DAOSD, and dose reduction of dupilumab may also be an effective treatment option. The pathomechanism of DAOSD is still not fully elucidated. In a prospective study low, but stable conjunctival GC numbers were observed in moderate-to-severe AD patients, before and during dupilumab treatment. However, the Mucin 5 AC (MUC5AC) expression of GCs decreased during dupilumab treatment, suggesting an impairment of the GC function by dupilumab treatment. In addition, higher dupilumab tear fluid levels were found in dupilumab-treated AD patients with moderate-to-severe OSD compared to patients with no or mild OSD, whereas the dupilumab serum levels are similar. Clinicians should be aware of the frequent occurrence of OSD in moderate-to-severe AD patients, and a low-threshold referral to an ophthalmologist is recommended

Quantification of therapeutic antibodies and endogenous proteins with LC-MS/MS

Therapeutic monoclonal antibodies are widely used for the treatment of various diseases were conventional small molecule based drugs are not effective. For patients with inflammatory autoimmune disease for example, the overproduction of TNF-alpha protein causes painful inflammatory symptoms. The level of TNF-alpha can be reduced through treatment with therapeutic monoclonal antibodies such as infliximab or adalimumab. However, some patients develop anti-drug antibodies that target these therapeutic ‘exogenous’ proteins thus reducing the drug concentration in plasma. This in turn renders the treatment ineffective. In this case quantitative proteomics is used for therapeutic drug monitoring (TDM) of drug concentration in plasma. This can provide the clinician useful insight to what is happening in the patient’s body and can be used to tailor the patient’s care. Quantitative proteomics can also be used for endogenous proteins. Endogenous proteins are synthesized by the DNA within the cell nucleus. Endogenous proteins are the building blocks for all cells that make up the organism, they play a role in food digestion, they offer protection from pathogens and they heal the cells when damaged. The ability to determine whether these proteins are present in the required concentration in plasma can help the clinician to diagnose a disease but also to personalize the treatment plan. Patients with hemophilia A for example, have a mutation in the X chromosome in locus q28. This portion of the X chromosome codes for the synthesis of the 2351 amino acid long FVIII protein. However, if FVIII is not synthesized correctly, this happens when one amino acid is swapped for another, misshaped or misfolded FVIII protein might be formed which could compromise the binding efficiency to its stabilizer protein von Willebrand or to the other factors FIX and FX involved in coagulation, resulting in prolonged bleeding times. Different dosing schemes with recombinant FVIII are thus incorporated dependent on the level of FVIII deficiency. This thesis provides a tutorial for quantitative proteomics with liquid chromatography− tandem mass spectrometry (LC-MS/MS), furthermore various examples of bioanalytical quantification of endogenous and therapeutic proteins in human plasma are presented. Novel sample purification strategies are introduced and optimized though experimental design. Some of these methods such as infliximab and adalimumab are already in use for routine TDM. Others, such as coagulation FVIII, neutralizing anti-drug antibodies, dinutuximab and active anti-thymocyte globulin (ATG) were developed for ongoing pharmacokinetics and pharmacodynamics studies

Quantification of therapeutic antibodies and endogenous proteins with LC-MS/MS

Therapeutic monoclonal antibodies are widely used for the treatment of various diseases were conventional small molecule based drugs are not effective. For patients with inflammatory autoimmune disease for example, the overproduction of TNF-alpha protein causes painful inflammatory symptoms. The level of TNF-alpha can be reduced through treatment with therapeutic monoclonal antibodies such as infliximab or adalimumab. However, some patients develop anti-drug antibodies that target these therapeutic ‘exogenous’ proteins thus reducing the drug concentration in plasma. This in turn renders the treatment ineffective. In this case quantitative proteomics is used for therapeutic drug monitoring (TDM) of drug concentration in plasma. This can provide the clinician useful insight to what is happening in the patient’s body and can be used to tailor the patient’s care. Quantitative proteomics can also be used for endogenous proteins. Endogenous proteins are synthesized by the DNA within the cell nucleus. Endogenous proteins are the building blocks for all cells that make up the organism, they play a role in food digestion, they offer protection from pathogens and they heal the cells when damaged. The ability to determine whether these proteins are present in the required concentration in plasma can help the clinician to diagnose a disease but also to personalize the treatment plan. Patients with hemophilia A for example, have a mutation in the X chromosome in locus q28. This portion of the X chromosome codes for the synthesis of the 2351 amino acid long FVIII protein. However, if FVIII is not synthesized correctly, this happens when one amino acid is swapped for another, misshaped or misfolded FVIII protein might be formed which could compromise the binding efficiency to its stabilizer protein von Willebrand or to the other factors FIX and FX involved in coagulation, resulting in prolonged bleeding times. Different dosing schemes with recombinant FVIII are thus incorporated dependent on the level of FVIII deficiency. This thesis provides a tutorial for quantitative proteomics with liquid chromatography− tandem mass spectrometry (LC-MS/MS), furthermore various examples of bioanalytical quantification of endogenous and therapeutic proteins in human plasma are presented. Novel sample purification strategies are introduced and optimized though experimental design. Some of these methods such as infliximab and adalimumab are already in use for routine TDM. Others, such as coagulation FVIII, neutralizing anti-drug antibodies, dinutuximab and active anti-thymocyte globulin (ATG) were developed for ongoing pharmacokinetics and pharmacodynamics studies

Pharmacokinetics in Elderly Women of Benzyl Alcohol From an Oil Depot

Pharmaceutical oil depots are meant to release active substances at a sustained rate. Most of these depots contain benzyl alcohol (BOH) to facilitate the production and administration. Because BOH changes the solubility of components in both the body fluid and the oil formulation, it is relevant to know the change in the BOH concentration in the oil over time. In this study, volunteers were subcutaneously injected with an oil depot that contained 10% BOH, nandrolone decanoate, and cholecalciferol. The aim of this study was to determine the pharmacokinetic profiles of BOH and its metabolites benzoic acid and hippuric acid simultaneously in serum to estimate the BOH release out of the depot. For this, an HPLC bioassay was developed and adequately validated. Hereafter, the bioassay was applied to serum samples obtained at several time points between 0 and 35 days. BOH appeared immediately in serum after injection. The pharmacokinetic profile revealed that all BOH was depleted from the depot within 52 h after injection. Thus, the partition coefficient of active substances between the oil formulation and the body tissue changes rapidly in the first days after injection but will remain constant hereafter

Quantification of active infliximab in human serum with liquid chromatography–tandem mass spectrometry using a tumor necrosis factor alpha -based pre-analytical sample purification and a stable isotopic labeled infliximab bio-similar as internal standard : A target-based, sensitive and cost-effective method

The therapeutic monoclonal antibody Infliximab (IFX) is a widely used drug for the treatment of several inflammatory autoimmune diseases. However, approximately 10% of patients develop anti-infliximab antibodies (ATIs) rendering the treatment ineffective. Early detection of underexposure to unbound IFX would result in a timely switch of therapy which could aid in the treatment of this disease. Streptavidin coated 96 well plates were used to capture biotinylated-tumor necrosis factor -alpha (b-TNF-α), which in turn was used to selectively extract the active form of IFX in human serum. After elution, IFX was digested using trypsin and one signature peptide was selected for subsequent analysis on liquid chromatography − tandem mass spectrometry (LC–MS/MS). The internal standard used was a stable isotopic labeled IFX bio-similar. The assay was successfully validated according to European Medicines Agency (EMA) guidelines and was found to be linear in a range of 0.5–20 μg/mL (r2 = 0.994). Lower limit of quantification for the assay (2 = 0.95 with a ρc = 0.83) and the accuracy was in line with previously published results. In conclusion, a sensitive, robust and cost-effective method was developed for the bio-analysis of IFX with LC–MS/MS by means of a target-based pre-analytical sample purification. Moreover, low volume and costs of consumables per sample promote its feasibility in (pre)clinical studies and in therapeutic drug monitoring. This method should be considered as first choice due to its accuracy and multiple degree of selectivity