Infliximab In Serum Research Articles

A Bottom-Up Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Infliximab: Method Development, Comparison With 2 Enzyme-Linked Immunosorbent Assay Methods, and Evaluation of Anti-Drug Antibody Interference.

Therapeutic drug monitoring is recommended to optimize infliximab use and improve outcome in chronic inflammatory disorders. To describe a simple and affordable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure infliximab in serum. Infliximab was measured using winged stable isotope-labeled peptides as internal standards. Linearity, lower limit of measuring interval, limit of detection, precision, accuracy, carryover, and ion suppression were evaluated. Method comparison against 2 enzyme-linked immunosorbent assay (ELISA) methods (Remsima Monitor and IDKmonitor Infliximab) and anti-drug antibody (ADA) interference were evaluated using clinical specimens from inflammatory bowel disease patients (N = 237). Analytical run time and sample preparation time were 5 minutes per sample and 3 hours per batch, respectively. Analytical measurement interval and limit of detection were 0.50 to 50.0 μg/mL (R2 = 0.998) and 0.25 μg/mL, respectively. The intraday and interday imprecision percentage coefficients of variation were less than 6.1%. Accuracy was 94.2% to 98.7%. No significant ion suppression or carryover was observed. Infliximab concentrations measured by LC-MS/MS showed good agreement with those measured by Remsima Monitor (mean percentage difference, 5.7%; 95% CI, -1.2% to 12.6%) but were markedly lower than those measured by IDKmonitor (-32.6%; -35.8% to -29.4%), demonstrating significant bias between ELISAs. Although a good agreement between LC-MS/MS and ELISA was observed for ADA-negative samples (-3.5%; -12.8% to 5.9%), a significant bias was observed for ADA-positive samples (13.6%; 1.7% to 25.6%). This simple, fast, and affordable LC-MS/MS method for infliximab quantitation could improve standardization of infliximab quantitation and optimization of infliximab use in patients with high-titer ADA.

Highly sensitive therapeutic drug monitoring of infliximab in serum by targeted mass spectrometry in comparison to ELISA data

BackgroundPresently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab.MethodsPeptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation.ResultsWe demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28–44% for peptide 1 and 64–97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides.ConclusionsIn this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates (n = 3–4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.

B-290 Development of a LCMS/MS Method for the Quantitation of Infliximab in Serum

Abstract Background Quantitative bottom-up proteomics of biotherapeutics has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. The general workflow includes multiple steps: sample extraction/purification, reduction, alkylation, enzymatic digestion, desalting, and LC-MS/MS analysis of signature peptides. In this study, we developed an infliximab drug assay with desired operational efficiencies needed in a clinical laboratory. Evaluations included reduction/alkylation removal, trypsin digestion length, trypsin quality, online/offline/no sample desalting, multiple/single signature peptides, and whole protein/peptide internal standardization. Methods For sample preparation, a general procedure was initially followed. Briefly, protein precipitation with ammonium sulfate, re-suspension with buffer, reduction, alkylation, overnight enzymatic digestion, desalting, and LC-MS/MS analysis. To simplify the workflow, the steps of reduction and/or alkylation were omitted. The trypsin digestion time was varied from 2 h, 4 h, and overnight. TPCK-modified trypsin from two different vendors (Thermo Scientific and Millipore Sigma) were also compared for digestion efficiency. SILu™MAb infliximab stable-isotope labeled monoclonal antibody (Millipore sigma) was compared with customer synthesized stable-isotope labeled signature peptides as the internal standard. Different trapping cartridges were evaluated for online sample desalting and different analytical columns were evaluated for target peptide analysis. A Transcend TurboFlow TLX UHPLC System coupled to a TSQ Altis triple-quadrupole mass spectrometer was used (Thermo Scientific) operating in multiple reaction monitoring mode (2 transitions for each peptide and internal standard). Two signature peptides YASE (Sequence: YASESMSGIPSR) and GLEW (Sequence: GLEWVAEIR) from different chains of infliximab were analyzed. Results For sample preparation, the omission of protein reduction/alkylation from the workflow did not significantly affect YASE quantitation. The peak area of GLEW decreased by about 30% without reduction/alkylation. The evaluation of three calibration curves corresponding to the digestion time of 2, 4, and overnight respectively showed that 4 h was optimal for all the levels of calibrators if both signature peptides were used for quantitation. TPCK-modified trypsin from two different vendors were equal in digestion efficiency. The calibration curve using SILu™MAb infliximab stable-isotope labeled monoclonal showed superior linearity (R2 > 0.98) and recovery for each calibrator vs using stable-isotope labeled signature peptides as internal standard. With the optimized conditions, both signature peptides can be used for the accurate quantitation of infliximab. However, for monitoring both peptides simultaneously, the chromatography separation needed 14.5 min. On the contrary, 9 min is needed for monitoring a single peptide YASE. Conclusion In summary, systematic optimization of sample preparation and LC-MS/MS analysis was accomplished in this study. The entire workflow was simplified by removing the steps of reduction and alkylation and using optimal digestion time, which increases the efficiency and significantly facilitate the implementation of this assay.

Simultaneous quantification and structural characterization of monoclonal antibodies after administration using capillary zone electrophoresis-tandem mass spectrometry

Monoclonal antibodies (mAbs) are demonstrating major success in various therapeutic areas such as oncology and the treatment of immune disorders. Over the past two decades, novel analytical methodologies allowed to address the challenges of mAbs characterization in the context of their production. However, after administration only their quantification is performed and insights regarding their structural evolution remain limited. For instance, clinical practice has recently highlighted significant inter-patient differences in mAb clearance and unexpected clinical responses, without providing alternative interpretations. Here, we report the development of a novel analytical strategy based on capillary zone electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for the simultaneous absolute quantification and structural characterization of infliximab (IFX) in human serum. CE-MS/MS quantification was validated over the range 0.4–25 µg·mL-1 corresponding to the IFX therapeutic window and achieved a LOQ of 0.22 µg·mL-1 (1.5 nM) while demonstrating outstanding specificity compared to the ELISA assay. CE-MS/MS allowed structural characterization and estimation of the relative abundance of the six major N-glycosylations expressed by IFX. In addition, the results allowed characterization and determination of the level of modification of post-translational modifications (PTMs) hotspots including deamidation of 4 asparagine and isomerization of 2 aspartate. Concerning N-glycosylation and PTMs, a new normalization strategy was developed to measure the variation of modification levels that occur strictly during the residence time of IFX in the patient’s system, overcoming artefactual modifications induced by sample treatment and/or storage. The CE-MS/MS methodology was applied to the analysis of samples from patients with Crohn’s disease. The data identified a gradual deamidation of a particular asparagine residue located in the complementary determining region that correlated with IFX residence time, while the evolution of IFX concentration showed significant variability among patients.

P453 The significance of the concentration of antibodies to infliximab on the frequency of loss of response when alternating the original drug infliximab and its biosimilar in patients with ulcerative colitis.

Abstract Background Biosimilars are analogues of biopharmaceutical drugs, with a close, but not identical, parent molecule. Biosimilars offer greater access to affordable treatment for inflammatory bowel disease, while being comparable in efficacy and safety to brand-name products. However, switching from original genetically engineered biological drugs (GEBD) to biosimilars requires study and analysis. Aim To determine the concentration of antibodies to infliximab in patients with UC receiving infliximab by one trade name (TN) and when alternating infliximab and its biosimilars and to determine the frequency of loss of response during the year of observation. Methods In the Department of inflammatory bowel diseases of the A. S. Loginov Moscow Clinical Scientific Center of the Moscow Healthcare Department, were observed 38 patients with IBD, who regularly received the original drug infliximab (IFX) -group 1 (n=20), group 2 a group of patients (n=18) alternated the original drug infliximab and its biosimilar.The level of antibodies to IFX in the blood serum before the next scheduled administration of the drug 10 months after inclusion in the study by enzyme immunoassay using the Shikari Quantitative Determination of Infliximab (Q-INFLIXI) and Shikari Quantitative Determination of Antibodies to Infliximab (Q-ATI) kits (Matriks Biotek). The loss of response was assessed by clinical, laboratory and instrumental data. Comparative analysis was carried out by the method of four-field tables using non-parametric statistical tests. Results Of the 20 patients with UC of the 1-st group, 3 (15.0%) had antibodies to IFX, on average, its concentration was 8.5 ± 2.2 ng/ml (normal values <5 ng/ml). Of the 18 patients of the 2nd group 10 (55.5%) antibodies to IFX were detected, on average - 187.9 ± 38.2 ng/ml. (OR - 0.141; 95% CI 0.030-0.658; x2 - 6.923, p= 0.023).During the year of observation among patients of the 1st group receiving the original IFX, the loss of response occurred in 2 (10.0%) patients with UC. Of the patients of the 2nd group, the loss of response occurred in 8 (10.0%) (RR - 0.139; 95% CI 0.025 - 0.785; x2 - 5.797; p = 0.017). Conclusion The concentration of antibodies to infliximab is higher in patients with UC when alternating IFX and its biosimilars than in patients with UC who regularly receive IFX by one TN. The frequency of loss of response when alternating infliximab and its biosimilars is significantly higher in patients with UC who regularly receive the drug for one TN.

P337 Therapeutic drug monitoring: standardization of Promonitor Quick IFX and Promonitor Quick ADL point of care tests with WHO International Standards for the quantification of infliximab and adalimumab in whole blood and serum

Abstract Background Promonitor Quick IFX and Promonitor Quick ADL are rapid point of care lateral flow tests (LFT) based on a sandwich immunoassay for the quantification of infliximab (IFX) and adalimumab (ADL), respectively, in human whole blood (finger prick or venous) or serum. These tests are to be used as an aid in Therapeutic Drug Monitoring (TDM) of inflammatory bowel disease (IBD) and rheumatic patients under anti-TNFα therapy. The international standards (IS) developed by World Health Organization (WHO) for IFX and ADL allow harmonization and comparability among different assays. The aim of this study, was to show that Promonitor Quick IFX and Promonitor Quick ADL can measure either reference or biosimilar drugs, as well as to evaluate the agreement of Promonitor Quick IFX and Promonitor Quick ADL tests and the WHO IS. Methods Clinical and Laboratory Standards Institute EP10-A3 guidelines were followed to estimate the bias of Promonitor Quick assays when used to quantify IFX or ADL in samples containing the reference drugs, biosimilars or the WHO IS. Briefly, whole blood was spiked with four known concentrations of IFX or ADL, including current clinical decision levels. Ten replicates were measured of each level along two days. Promonitor Quick IFX was evaluated using the reference drug, SB2 and CT-P13 biosimilars, and the WHO IS (NIBSC, 16/170). Promonitor Quick ADL was evaluated using the reference drug, ABP501 and SB5 biosimilars, and the WHO IS (NIBSC, 17/236). Results were obtained in combination with the automated portable reader PQreader. Results Bias was estimated by comparing the observed concentration of drug spiked whole blood samples. Each biosimilar was compared to the reference at the different drug levels tested. Results showed that Promonitor Quick IFX (Table, 1) and Promonitor Quick ADL (Table, 2) are able to measure equivalently any molecule. The accuracy or closeness of the agreement between the result provided by Promonitor Quick IFX and Promonitor Quick ADL and the true value of the measurand was assessed by measuring the IS developed by the WHO (Tables, 1 & 2). Similar results were obtained when serum matrix was used. Conclusion Promonitor Quick IFX and Promonitor Quick ADL allow monitoring of patients treated with IFX and ADL, respectively, with just a finger prick sample. Both tests can quantify reference and biosimilar drugs with equivalent results. Moreover, comparable results were obtained with the WHO IS, and thus, demonstrate that Promonitor Quick tests are suited to accurately determine drug levels at the clinical decision points in both whole blood and serum, proving to be an effective and valuable tool in TDM and immediate decision making in the doctor office or hospitals.

P384 Therapeutic drug monitoring: performance of the first lateral flow-based Point of Care test for the quantification of infliximab in a finger prick

Abstract Background Promonitor Quick IFX is a lateral flow test (LFT) for the quantification of infliximab (IFX) in human whole blood (finger prick or venous) or serum in 20 minutes. This LFT is based on a sandwich immunoassay to quantify either the reference IFX or biosimilars. This abstract describes the studies performed to establish the analytical specifications of the product. Methods Clinical and Laboratory Standards Institute (CLSI) guidelines were followed for the evaluation of the analytical specifications of the LFT in whole blood and serum matrices: Linearity (EP-06-A), Detection capability (EP17-A2), Interfering substances (EP07, 3rd Edition), Intermediate precision (EP05-A3) and Bias evaluation study for Biosimilars (EP10-A3). Results were obtained in combination with the automated portable reader PQreader. A Datamatrix provided with each Promonitor Quick IFX kit contains the calibration information required for the PQreader to measure the Control and Test lines and report the IFX concentration. Results The linear assay range was determined to be 1-58 µg/mL in whole-blood and 0.6-67 µg/mL in serum according to the processes indicated in the Package Insert. The Limit of Blank is 0.8 μg/mL, the Limit of Detection and Lower Limit of Quantification (LLoQ) are 1.1 μg/mL, and the Upper Limit of Quantification (ULoQ) is 15.4 μg/mL. There was no effect on assay performance when each of the following substances were added to samples with 0, 3, and 7 μg/mL of IFX: Haemoglobin (>1000 mg/dL), Bilirubin (>40 mg/dL), Triglycerides (>1500 mg/dL), HAMA (160 AU/mL), Rheumatoid factor (200 IU/mL), EDTA (5.4 mg/mL), Heparin (51 U/mL), Citrate (11.4%), Vedolizumab (60 μg/mL) and Adalimumab (20.25 μg/mL). Repeatability and within-device precision results obtained for the positive samples are shown in the table below. The negative samples showed a negative result in all the measurements. A bias study showed that Promonitor Quick IFX can quantify CT-P13, SB2 and GP1111 biosimilars throughout the measurement range with a maximum bias of 14%. Conclusion Promonitor Quick IFX is the first LFT available for true Point of Care testing of patients treated with IFX with just a finger prick sample. It provides quick turnaround time to facilitate therapeutic drug monitoring and aid immediate decision making in the doctor office or hospitals with an excellent analytical performance.

Dynamics of serum concentrations of antibodies to infliximab: a new approach for predicting secondary loss of response in inflammatory bowel diseases.

Background:Antibodies to infliximab (ATI) in serum are associated with secondary loss of response (LOR) to infliximab (IFX) therapy in patients with inflammatory bowel disease (IBD). However, feasible ATI-related predictors of therapy success are lacking and knowledge about individual ATI dynamics is limited. Therefore, this study analyzed whether ATI dynamics are able to predict LOR to IFX therapy and compared their predictive power with known predictors of LOR to IFX.Methods:This was a retrospective study of patients with Crohn’s disease (CD) or ulcerative colitis (UC) on IFX maintenance therapy and proactive IFX and immunogenicity monitoring in an outpatient clinic in Germany. Slopes of ATI (SATI) and IFX levels (dynamic parameters) and medians of ATI, IFX, C-reactive protein, and fecal calprotectin (static parameters) were calculated over a defined period of time after ATI emergence. Dynamic and static parameters were analyzed for associations with end points infliximab discontinuation due to secondary LOR and total IFX discontinuation.Results:In all, 500 visits from 38 IBD patients (28 CD, 10 UC) with a median IFX maintenance duration of 68.2 weeks were evaluated. Grouping by SATI (ATI-N = ATI nondetectable, ATI-↓ = negative SATI, ATI-↑ = positive SATI) yielded significant differences for outcomes LOR (p = 0.004) and total IFX discontinuation (p = 0.01). Patients in the ATI-↓ group survived significantly longer LOR-free compared with the ATI-↑ group (p = 0.02). Cox regression confirmed SATI to be a significant risk factor for LOR (p = 0.002). An SATI cut-off of approximately 2.0 AU mL−1 week−1 was determined to predict LOR with 83.3% sensitivity and 93.8% specificity.Conclusion:The ATI slope-based index SATI is a new feasible diagnostic predictor of LOR in IBD patients. SATI may facilitate quick therapeutic decisions after ATI emerge.

Simultaneous Quantification of Free Adalimumab and Infliximab in Human Plasma Using a Target-Based Sample Purification and Liquid Chromatography-Tandem Mass Spectrometry.

Therapeutic drug monitoring of tumor necrosis factor alpha (TNF-α) inhibitors such as adalimumab (ADM) and infliximab (IFX) is considered of added value for patients with systemic inflammatory diseases. In contrast to enzyme-linked immunosorbent assay methods, liquid chromatography-tandem mass spectrometry methods allow for simultaneous quantification of multiple target antibodies in 1 run and thus providing a higher sample throughput. We describe a fast sample work-up strategy for the absolute and simultaneous quantification of ADM and IFX therapeutic monoclonal antibodies in human plasma samples using a target-specific sample purification in combination with liquid chromatography-tandem mass spectrometry. The sample purification was based on the selective capture of ADM and IFX in human plasma or serum using biotinylated TNF-α (b-TNF-α), which was coated on a streptavidin 96-well plate. After elution, analytes were heat denatured and trypsin digested to obtain signature peptides for quantification. Stable isotopically labeled ADM and IFX were introduced as internal standard before sample purification. The method was successfully validated following current European medicines agency guidelines. The linear dynamic rage for both analytes were 1-32 mcg/mL with an excellent mean coefficient of determination, R = 0.9994 for ADM and 0.9996 for IFX. Within-run and between-run imprecision and accuracy were within acceptance criteria. Cross-validation against enzyme-linked immunosorbent assay method showed a high between-method correlation R = 0.962 for ADM and R = 0.982 for IFX. This method provides an easy, efficient, and cost-effective workflow for therapeutic drug monitoring patients treated with ADM or IFX.

Inductively coupled plasma mass spectrometry assay for quantification of free infliximab in serum

TNF antagonists such as infliximab are effective for the treatment of several inflammatory and autoimmune diseases. Recent clinical studies have advocated the importance of measuring trough infliximab levels to guide treatment decisions. We have developed a novel assay for measuring serum free infliximab levels using inductively coupled plasma-mass spectrometry (ICP-MS). The method involves the incubation of patient serum in wells coated with recombinant TNF, followed by detection with lanthanide-labeled monoclonal anti-human IgG1 and ICP-MS analysis. Full method validation was performed and results for clinical samples tested with the new method were compared with those obtained from a capture ELISA and a cell-based assay. Validation of the ICP-MS assay revealed a lower limit of detection of 0.4 μg/mL in serum. The linear range of quantitation was 1–50 μg/mL. The within-run and between-run precision had a coefficient of variation (CV) of <10%, and the accuracy of the assay had a CV of <15%. In serum samples, the ICP-MS method was devoid of analytical interferences by high levels of hemoglobin, bilirubin and triglycerides. Serum sample results from 123 drug-naïve donors revealed a test cutoff at 0.5 μg/mL. Test results from clinical samples obtained by the ICP-MS method showed strong correlation with both the ELISA and cell-based assay. The ICP-MS methodology presented in this study is a robust method for measuring TNF antagonist serum levels, which makes it well suited for therapeutic drug monitoring in the clinical laboratory.

An automated mass spectrometric blood test for therapeutic drug monitoring of infliximab

Infliximab is a monoclonal antibody therapy used to treat several chronic immune-mediated diseases, including Crohn’s disease, ulcerative colitis, and rheumatoid arthritis. Infliximab acts by binding to tumor necrosis factor and, thus, inhibiting the inflammatory cascade. While it is a highly effective therapy, a subset of patients on infliximab will develop a loss of response to therapy. In these circumstances, therapeutic drug monitoring of infliximab offers a rational approach to clinical decision making and is associated with improved outcomes. While infliximab has most commonly been measured by immunometric approaches, mass spectrometric approaches offer the opportunity to improve test accuracy and reduce test costs. Herein, we describe a simple, bottom-up high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) approach for quantitation of infliximab in serum. Method development included pre-digestion and digestion experiments to determine critical sample preparation steps, optimization of the workflow and selection of rapidly produced proteolytic peptide(s) for quantitation. The workflow was further improved by automating all sample preparation steps on a robotic liquid handler, facilitating implementation in routine clinical use. A method comparison was performed against a Health Canada and US Food and Drug Administration licensed enzyme-linked immunosorbent assay. Our LC–MS/MS assay accurately reported concentrations based on drug manufacturer targets and demonstrated no interference from endogenous antibodies to infliximab; immunoassay methods did not share these performance characteristics. This LC–MS/MS method provides a workflow amenable to implementation in a clinical laboratory and desired performance characteristics for guiding clinical decision making.

Verification between Original and Biosimilar Therapeutic Antibody Infliximab Using nSMOL Coupled LC-MS Bioanalysis in Human Serum

Background: Infliximab (IFX) is a chimeric therapeutic monoclonal antibody targeting tumor necrosis factor alpha (TNFα)-mediated inflammatory immune diseases. However, despite of an initial good clinical response, decrease in response to long-term treatment is a common observation.Objective: Recent studies suggest that IFX level in circulation has a correlation with clinical bioavailabil-ity. Therefore, the management of IFX dosage for individual manifestation by IFX monitoring may be valuable for the improvement of therapeutic response and outcomes.Method: In order to develop a broad IFX therapeutic monitoring in human serum, we have developed the validated IFX bioanalysis for RemicadeTM and its biosimilar product using our nano-surface and molecu-lar-orientation limited proteolysis (nSMOL) technology coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The nSMOL chemistry has a unique property of Fab-selective prote-olysis, and makes it possible a global bioanalysis for many monoclonal antibodies.Results: The quantitation range of IFX in serum was from 0.293 to 300 μg/ml with good linearity. Quan-titation verification at the concentrations of 0.293, 0.879, 14.1 and 240 μg/ml was within 1.56-7.53% of precision and 98.9-111% of accuracy using H-chain signature peptide SINSATHYAESVK. Moreover, cross-verified bioanalysis of Remicade quantitation using biosimilar standard, and its opposite combina-tion, obtained an identical and inter-comparative results.Conclusion: The nSMOL strategy has the potential as a practical therapeutic monitoring technology in IFX therapeutic applications.

Quantitation of Infliximab and Detection of Antidrug Antibodies in Serum by Use of Surface Plasmon Resonance.

Monitoring infliximab (IFX) concentrations and the presence of antidrug antibodies (ADA) is important for patient management. We developed a method to measure IFX and ADA in serum in a single injection using surface plasmon resonance (SPR). Using the Bio-Rad ProteOn XPR36, tumor necrosis factor-α and IFX were covalently immobilized onto separate lanes of a chip surface. Diluted serum was injected over both lanes, followed by an injection of goat antihuman antibody. The binding response was used to quantify IFX or detect ADA. The analytical performance of the assay was determined. Using 50 patient samples, SPR results were compared with results from a reporter gene assay (RGA). For the quantification of IFX, the functional sensitivity was 0.5 μg/mL. The total precision was <10% for all concentrations tested. IFX concentrations measured by SPR correlated well with RGA (R = 0.862), but a bias was observed (slope = 0.61). SPR detected 14 ADA-positive samples. Compared with RGA for ADA detection, there were 6 true-positive, 8 false-positive, 5 false-negative, and 31 true-negative findings. SPR can be used to measure biological drug concentrations and detect ADA in serum. This technique may provide complementary information to current methods used to detect ADA.

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 0.5–20μg/mL (r2=0.994). Lower limit of quantification for the assay (<20% CV) was 0.5μg/mL, requiring only 2μL of sample. Cross-validation against enzyme-linked immunosorbent assay (ELISA) resulted in a high correlation between methods (r2=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.

Fiber optic-SPR platform for fast and sensitive infliximab detection in serum of inflammatory bowel disease patients

Infliximab (IFX) is a therapeutic monoclonal antibody used for treating patients with inflammatory bowel disease (IBD). In order to improve therapeutic outcomes it is recommended to monitor IFX trough concentrations. Although ELISA is currently widely used for this purpose, this method is not suitable for single patient testing. In this paper we describe the development of a fast bioassay for determining IFX concentration in serum using an in-house developed fiber-optic surface plasmon resonance (FO-SPR) biosensor. Studies were first conducted to optimize covalent immobilization of the IFX-specific antibody on the sensor surface as well as to select an optimal blocking buffer for restraining the non-specific binding. In order to reach clinically relevant sensitivity for detecting IFX in patients' serum, the SPR signal was amplified by employing gold nanoparticles functionalized with another set of IFX specific antibodies. Using the optimized sandwich bioassay, calibration curves were made with series of IFX concentrations spiked in buffer and 100-fold diluted serum, reaching the limit of detection of 0.3 and 2.2ng/ml, respectively. The established bioassay was finally validated using five IFX treated IBD patients samples. Results from the FO-SPR platform were compared with an in-house developed, clinically validated ELISA resulting in excellent Pearson and intraclass correlation coefficient of 0.998 and 0.983, respectively. Furthermore, the assay time of the FO-SPR platform was significantly reduced compared to ELISA, demonstrating the potential of this platform to be used as a point-of-care diagnostic tool for improving therapeutic outcomes of IBD patients.

Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC–MS/MS

ObjectivesAlthough therapeutic concentrations of infliximab (chimeric IgG1 kappa) are associated with improved clinical prognosis, clinical laboratory methods for monitoring are limited. Therefore, we aimed to develop a LC–MS/MS method to measure infliximab in serum. MethodsInfliximab was measured using isotope-labeled peptides and horse IgG as a surrogate internal standard. After trypsin digestion, peptides were separated by reverse-phase C8 LC and detected by MS/MS on an ABSciex API 5000; analyte-to-internal standard peak area ratios were used for quantitation. Sera from patients receiving infliximab were collected at different time points in treatment and compared with a commercially available ECLIA method. ResultsThe linear dynamic range of the assay was 1–100μg/mL (R2>0.998); both intra- and inter-assay imprecisions were <20%. Patients undergoing infliximab therapy had trough concentrations of 8.5±8.8μg/mL (mean±SD), which substantially increased 48–72h after infusion (77±40μg/mL), then fell after 28–32days (15±11μg/mL). A comparison of LC–MS/MS and ECLIA methods demonstrated a slope of 0.967 (95% CI: 0.894–1.034, r=0.970). ConclusionsWe have demonstrated the ability to quantify infliximab in patients using clonotypic peptides. This approach has the potential to be quickly adaptable to other monoclonal antibodies and to expand the availability of testing for this class of therapeutics in routine clinical practice.

Tu1292 Quantification of the Concentration of Antibodies Against Infliximab (IFX) in Human Serum Using a Pure Antibody As Calibrator

Tu1292 Quantification of the Concentration of Antibodies Against Infliximab (IFX) in Human Serum Using a Pure Antibody As Calibrator

Development and Validation of LC–MS/MS Method for the Quantitation of Infliximab in Human Serum

Liquid chromatography tandem mass spectrometry (LC–MS/MS), especially triple-quadrupole mass spectrometry, has become an attractive alternative method to ligand binding assays for therapeutic monoclonal antibody quantification in biological samples, but the use of an internal standard with infliximab LC–MS/MS assays has not been reported yet. In this study, an improved LC–MS/MS method for quantification of infliximab in human serum was developed and validated. A surrogate peptide was used as a representative of infliximab which was cleaved for the quantification of infliximab based on LC–MS/MS assay. A stable isotope-labeled signature peptide was used as the internal standard (IS). The results showed linearity in the range of 0.39–100 μg mL−1; the lower limit of quantification (LLOQ), and the lower limit of detection were 0.39 and 0.0975 μg mL−1, respectively. The quality control (QC) data showed that the within-run, between-run precision (%RSD) and accuracy (%RE) conformed to the acceptance criteria of ±15 % for calibration standards and QCs (±20 % at the LLOQ). Other validation parameters including selectivity, methanol precipitation efficiency, serum matrix effect, stability, and auto-sampler carry-over were also evaluated. This improved LC–MS/MS method might be a promising LC–MS-based methodology for pharmacokinetic studies of other recombinant monoclonal antibodies.

P020. Quantification of the concentration of antibodies against Infliximab in human serum using a pure antibody as calibrator

P020. Quantification of the concentration of antibodies against Infliximab in human serum using a pure antibody as calibrator

A generic sample preparation approach for LC–MS/MS bioanalysis of therapeutic monoclonal antibodies in serum applied to Infliximab

In this study, we developed a generic bioanalytical workflow providing sensitive, specific, and accurate absolute quantification of therapeutic monoclonal antibodies in serum. The workflow involves magnetic beads coated with protein A to pull-down therapeutic monoclonal antibodies with affinity for protein A from the biological matrix, followed by tryptic digestion and LC-MS/ MS quantification of a unique signature peptide, considering of course the matrix of interest and other present mAbs, if applicable. The feasibility of this approach was demonstrated for Inflix imab (trade name Remicade) in rat serum. The assigned signature peptide was monitored in the selected reaction monitoring (SRM) mode. Assay variability was determined to be below 20%, except at the QC low level, which was provided through optimization of the sample preparation and monitoring of the LC-MS/MS using a stable isotope labeled signature peptide as internal standard. The 100 ng/ml lower limit of quantification using only 25 µl sample volume, is generally considered as sufficient for pharmaceutical development purposes for monoclonal antibodies.