The European Bioanalysis Forum discusses a recommended shift from the traditional 3-Tier immunogenicity testing approach to a more adaptive immunogenicity testing strategy for biotherapeutics, emphasizing clinical relevance, risk-based considerations, assay performance, and collaboration with regulatory authorities to improve the assessment of anti-drug antibodies in therapeutic contexts. The recommendations encourage transitioning toward adaptive 1-Tier or 2-Tier combined approaches tailored to the molecule context-of-use. The new strategy prioritizes the clinical impact of anti-drug antibodies over mere incidence, advocating careful consideration of assay characteristics during assay development. The European Bioanalysis Forum continues to call for published case studies, industry cooperation, and proactive dialogue with regulatory authorities to challenge the traditional 3-Tier approach.

BACKGROUND:Pharmacokinetic research of infliximab often relies on prospectively collected, long-term frozen serum samples. Although the effect of storage conditions on infliximab stability has been researched extensively, the influence of long-term storage on the reliability of infliximab concentration measurements remains unknown. METHODS:In this proof-of-concept study, infliximab serum concentrations measured after 2, 5, and 8 years of standardized frozen storage at -70 °C were compared with paired samples analyzed directly after collection. Equivalence was assessed using a two one-sided tests framework with predefined margins of 80%-125%, in line with FDA equivalence guidance. RESULTS:Equivalence was demonstrated at all three storage durations. Geometric mean ratios were 87.0% (90% CI, 80.9%-93.6%), 103.2% (90% CI, 90.3%-117.9%), and 99.3% (90% CI, 92.5%-106.6%) at 2, 5, and 8 years, respectively, all within predefined equivalence margins. A statistically significant reduction was observed in the 2-year group (P = .010), though within equivalence bounds. No concentration-dependent bias was detected (P = .926), and Spearman correlation between fresh and frozen concentrations was high (ρ = 0.908, P < .001). CONCLUSION:Infliximab serum concentrations remain stable after long-term frozen storage of up to 8 years under standardized conditions, supporting the validity of deferred concentration analyses in prospectively stored serum samples.

Using a real-world psoriasis cohort, we established the pharmacokinetic-pharmacodynamic (PKPD) relationship for the biologic therapy adalimumab and evaluated the clinical utility and cost-effectiveness of a proactive therapeutic drug monitoring (TDM) strategy. A total of 543 patients on adalimumab monotherapy for psoriasis provided 946 pharmacokinetic samples and 1700 Psoriasis Area Severity Index (PASI) disease severity measurements. To describe the PASI change over time, a one-compartment linear PK model with first-order absorption and elimination was linked to a turnover mechanism of skin lesions. Based on the PKPD relationship and a predefined therapeutic range, real-time stochastic simulation was performed for a proactive TDM strategy, where trough levels guided dose escalation or reduction. Compared to the standard care, the TDM strategy improved PASI90 and PASI75 by 37.5% and 12.8%, respectively, with a 25.9% increase in drug costs. In the future, incorporating the PKPD model into a Bayesian therapeutic monitoring algorithm could facilitate individualized adalimumab dosing.

Antibodies produced by B cells aid in the recognition and clearance of pathogens and are the cornerstone of vaccination strategies. Humans produce nine different antibody isotypes, and their effector functions differ according to the type of antigen and route of exposure. Phenotypic variation between isotype-switched B cell subsets is expected but not studied in detail. To obtain a molecular definition of isotype-defined cell identity, we performed proteomics and transcriptomics on isotype-defined populations of human naive and memory B cells (MBCs): CD27-IgM+IgD+, CD27+CD38lo/-IgM+IgD+, CD27+CD38lo/-IgM+IgD-, and IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 MBCs (CD27+CD38lo/-Ig+). Combined proteome and transcriptome analysis revealed that mRNA and protein expression profiles separate isotype-defined B cell subsets according to their differentiation status. mRNA and protein expression levels correlated reasonably well for many genes. IgG4-switched B cells were most distinct from naive B cells in terms of mRNA as well as protein expression profiles. Besides a distinct expression profile of cytokine and Fc receptors, we identified a high expression of IgE-coding mRNA in IgG4-switched B cells. SDR16C5 was identified as uniquely upregulated in IgG4-switched B cells. Taken together, this study highlights the distinct phenotypic profile of IgG4-switched B cells.

Antibody responses to individual antigens vary widely in their relative contributions of the four human IgG subclasses. Detection of IgG subclasses is usually done using specific monoclonal antibodies. Ideally, such antibodies recognize a subclass irrespective of polymorphic structural variation (also called allotypes), while not recognizing any of the other subclasses. For IgG2 this is especially challenging. There are many monoclonal antibodies commercially available, but whether they meet the abovementioned requirements is unclear. In this study, we examined twelve commercially available monoclonal antibodies against human IgG2 (HP6002/AB4-DH4, HP6014/AC3-AA11/MH162.1, HP6200/HG2-56F, KT138, 985028/MAB9794, 31-7-4, MTG211E, 52G1, EPR4418, 2348B/MAB97941, and RM118) for their ability to selectively bind to human IgG2, and not to the other three subclasses. We used a panel of recombinant monoclonal antibodies comprising different human IgG subclasses and allotypes, and tested binding in ELISA. Of the twelve anti-human IgG2 clones tested, only three were found to be highly specific for IgG2 (>1000-fold). Two further clones demonstrated good selectivity albeit with measurable cross-reactivity to IgG4 of up to ca. 1-2%. Three clones were cross-reactive to certain IgG4 and IgG3 allotypes. These clones may possess G4m(b) specificity. The remaining clones showed only low selectivity for IgG2. In conclusion, specificity determinations of anti-IgG subclass reagents should take into account the genetic/allotypic variation across IgG subclasses. Antibodies with proclaimed IgG2 specificity do not always meet requirements for subclass-specific antibody detection.

After antigen encounter, long-lived antibody-secreting cells (ASC) secrete high-affinity circulating antibodies. In addition, memory B cells (MBC) are quickly reactivated upon antigen re-exposure and predominantly generate shorter-lived ASCs. Studies have suggested that MBC can differentiate into ASCs without recognizing their cognate antigen, a process known as “bystander activation”. This antigen-independent reactivation of MBC could help maintain circulating antibody levels, thereby protecting against future infections. To elucidate whether SARS-CoV-2 mRNA vaccination leads to bystander activation of B cells, the dynamics of antibody concentrations against six pathogen-specific antigens not encountered during the sampling period were analyzed over time. Deep profiling of antigen-specific B cell responses was simultaneously performed using multiparameter high-dimensional spectral flow cytometry. Antibody concentrations against tetanus toxoid (TT), respiratory syncytial virus (RSV), and influenza hemagglutinin (HA) unexpectedly increased 6 weeks after the first SARS-CoV-2 vaccination. Deep profiling of B cell differentiation stages demonstrated a short-term increase in influenza-specific IgG+ DN3 B cells, RSV-specific IgG+ CD11c+ activated B cells, and TT-specific IgG+ MBC following vaccination. In this study, we demonstrated at both the antibody and cellular levels that SARS-CoV-2 mRNA vaccination transiently activates distinct early activated B cell compartments directed against influenza HA, RSV, and TT.

Detection and characterization of antigen-specific T cells are important for studying immune responses upon infection, vaccination, or autoreactivity. The activation-induced marker (AIM) assay is a robust technique to identify and characterize antigen-specific CD4 and CD8 T cells. However, there is variability in the AIM assay, particularly in the type and number of activation markers used. In this study, we set out to define which marker combinations are most suited to optimally detect antigen-specific CD4 and CD8 T cells and if certain marker combinations preferentially detect specific CD4 T helper subsets. A multiparameter flow cytometry panel, including six common activation markers: CD40L, CD137, CD69, OX40, CD25, and PD-L1, was used for detecting antigen-specific T cells following infection (SARS-CoV-2 and CMV) or vaccination (mRNA-1273 SARS-CoV-2). We demonstrate that combining multiple activation markers increases the detection frequency of antigen-specific CD4 T cells compared to commonly used dual marker combinations. In addition, marker combinations including PD-L1 detected a higher frequency of antigen-specific CD4 T cells in SARS-CoV-2 and CMV infected but not in SARS-CoV-2-vaccinated individuals. Certain dual marker combinations preferentially detected specific CD4 T helper subsets. The majority of antigen-specific CD8 T cells were captured by the dual combination of CD69 plus CD25. In conclusion, combining CD137, CD69, OX40, CD25, and PD-L1 in an AIM assay results in robust and optimal detection of both specific CD4 T helper subsets and CD8 T cells in different antigenic contexts.

BackgroundTherapeutic proteins such as adalimumab can elicit an antibody response. How dosing regimens impact immunogenicity remains ill-understood, especially with respect to the frequency of dosing.ObjectiveWe aimed to investigate the relationship between single versus multiple adalimumab doses and immunogenicity, in terms of anti-drug antibody production and skewing towards the non-inflammatory immunoglobulin G4 (IgG4) subclass.MethodsImmunoglobulin M, immunoglobulin G, and IgG4 anti-drug antibodies were analyzed in retrospective cohorts of healthy individuals and patients with rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, or psoriasis, receiving one or multiple doses of adalimumab, using optimized drug-tolerant assays, newly developed in the case of IgG4.ResultsA single dose of adalimumab proved highly immunogenic, while repeated dosing, resulting in prolonged exposure to high antigen concentrations, led to attenuation of the anti-drug antibody response. Immunoglobulin G4 anti-drug antibodies developed earlier than previously reported with drug-sensitive assays, appearing as early as week 3, even after a single dose of adalimumab. Skewing towards IgG4 was nevertheless stronger with repeated dosing.ConclusionsRepeated high-dose adalimumab exposure can limit both the magnitude and inflammatory potential of the antibody response. These results highlight drug exposure as a factor modulating the immunogenicity of biologics.

BACKGROUND:Extended interval dosing (EID) of natalizumab is increasingly used in clinical practice for patients with multiple sclerosis, driven by growing evidence supporting its sustained efficacy and associated benefits. However, it remains unclear whether EID, particularly at dosing intervals beyond six weeks, affects treatment satisfaction. OBJECTIVES:To investigate whether natalizumab dosing intervals of up to ten weeks are associated with patient-reported treatment satisfaction. METHODS:Participants from the prospective NEXT-MS and SUPERNEXT trials, both evaluating concentration-guided EID while preserving therapeutic drug concentrations, completed longitudinal Treatment Satisfaction Questionnaire for Medication (TSQM) assessments. Associations between dosing interval and scores in the four TSQM domains (Effectiveness, Side effects, Convenience, and Overall satisfaction) were analyzed using linear mixed-effects models. RESULTS:A total of 427 participants were included. Most participants reported high treatment satisfaction. No overall association was observed between dosing interval and TSQM scores. However, additional effect-modification analyses suggested that the association between dosing interval and treatment satisfaction differed according to treatment duration, disease duration, baseline EDSS, and route of natalizumab administration. CONCLUSIONS:Treatment satisfaction was high and was not affected by longer dosing intervals in the majority of patients. However, additional effect-modification analyses indicated that the impact of dosing interval on treatment satisfaction may differ according to patient characteristics, highlighting the importance of individualized treatment decisions.

Natalizumab, a monoclonal antibody used to treat multiple sclerosis, can undergo Fab-arm exchange (FAE) with endogenous IgG4, resulting in a monovalent form with reduced avidity and presumed lower potency than the parental bivalent form. Levels of bivalent and monovalent natalizumab depend on the ratio of endogenous IgG4 to total natalizumab, which varies substantially between individuals. The clinical relevance of natalizumab FAE remains unknown. This study investigated the potential clinical implications of bivalent and monovalent natalizumab levels regarding progressive multifocal leukoencephalopathy (PML), extended interval dosing (EID) and wearing-off symptoms, using multiple cohorts of natalizumab-treated patients. Seven natalizumab-associated PML cases were each matched to two controls. Contrary to the hypothesis that higher bivalent levels might increase PML risk, cases showed a trend toward lower bivalent natalizumab levels, although definite conclusions were limited by small sample size. In patients receiving EID, both total and bivalent natalizumab levels decreased compared to standard interval dosing, with a greater median individual decrease in bivalent natalizumab than in total natalizumab (94% versus 78%). Notably, in more than half of patients receiving EID, bivalent natalizumab levels fell below the quantification limit (<0.1 µg/mL). Finally, patients experiencing wearing-off symptoms during natalizumab treatment had lower bivalent natalizumab levels, although this warrants validation in larger cohorts.

Introduction : In 2023, the natalizumab biosimilar Tyruko® was approved for relapsing-remitting multiple sclerosis. Here, we assessed patient experiences and natalizumab serum concentrations in a real-world cohort of patients who switched from the originator, Tysabri®, to the biosimilar. Furthermore, we evaluated the consistency of the new John Cunningham virus (JCV) assay that comes with the biosimilar. Methods : Patients, aware of the switch, completed questionnaires and had natalizumab concentrations measured during treatment with both products. Questionnaires assessed impact of MS symptoms, treatment satisfaction and wearing-off symptoms. An additional questionnaire assessed perceived differences or side effects with the biosimilar. JCV results of the new ImmunoWELL assay were analyzed over time. Results : Among 83 patients, 15% reported more side effects during treatment with the biosimilar. Overall satisfaction was lower during treatment with the biosimilar compared to the originator. Other questionnaire scores and trough concentrations did not differ significantly. JCV results of the ImmunoWELL assay remained mainly consistent. Conclusion : While most patients perceived the biosimilar as equivalent to the originator, overall satisfaction was lower, and some reported more side effects. As these findings differ from blinded approval trials, a nocebo effect may influence patient perceptions in real-world settings when switching from originator to biosimilar natalizumab.

In humans, the stages and dynamics of B cell development after antigen encounter remain unclear. Identifying early B cell differentiation stages could reveal biomarkers for humoral immunity and potential targets to prevent unwanted antibody responses. We characterized antigen-specific B cell responses longitudinally after SARS-CoV-2 mRNA vaccination using multiparameter spectral flow cytometry. Spike-specific IgG+ CD27+ CD71+ activated B cells (ActBCs), presumed to be germinal center-derived and IgG+ DN2 extrafollicular B cells, dominated the early antigen-specific B cell response, while memory B cells were the main population 6 months after vaccination. Within the IgG+ ActBC compartment, we delineated six novel clusters with specific contraction dynamics. Following the second vaccination, certain ActBC clusters displayed sustained expansion over time, being phenotypically similar to memory B cells, while others strongly expanded and subsequently contracted. Several of the rapidly contracting ActBC clusters expressed CD11c, a defining marker for atypical B cells, suggesting a possible extrafollicular origin of these clusters. The transient presence of heterogeneous ActBC clusters was also observed for total B cells when gated in an antigen-independent manner. Characterization of novel ActBC clusters early after antigen encounter helps delineate and dissect the complexity of B cell differentiation, which is vital for understanding unwanted B cell responses.

OBJECTIVE:Rheumatoid arthritis (RA) and Sjögren's disease (SjD) are two rheumatic autoimmune diseases that commonly co-occur. Besides some overlap in clinical presentation, the two diseases share the frequent occurrence of rheumatoid factors (RF); autoantibodies targeting the constant region of IgG (IgG-Fc). We previously demonstrated that RFs of patients with RA or SjD have distinct reactivity profiles towards various epitopes on IgG-Fc, prompting the question of whether binding patterns in patients with RA-associated SjD (SjD-RA) would reflect either disease or a combination thereof. METHODS:We determined IgM- and IgA-RF levels against a selected set of RF targets that were developed to map preferential RF binding to specific epitopes. We assessed binding patterns among 14 patients with SjD-RA and compared these to patterns found among 28 patients with early RA and 152 patients with SjD. RESULTS:Both IgM-RF and IgA-RF profiles of SjD-RA patients closely resemble those of RA patients and are distinctly different from those found among SjD patients. Furthermore, the presence and binding pattern of RF seems to be inherently linked to the presence of anti-SSA/Ro antibodies in patients with SjD. CONCLUSIONS:Analysing RF binding patterns could help to distinguish SjD, SjD-RA, RA and contributes to a better understanding of the underlying immunological processes in a complex multidisorder setting.

Background Natalizumab is a highly effective drug for patients with relapsing-remitting multiple sclerosis (MS). A disadvantage of this treatment is the risk of progressive multifocal leukoencephalopathy in patients who are seropositive for the John Cunningham virus (JCV). JCV seroconversion rates increase under natalizumab treatment compared with non-natalizumab using controls. The aim of this study was to assess whether lower natalizumab trough concentrations are associated with reduced JCV seroconversion compared with higher natalizumab trough concentrations. Methods Two overlapping cohorts of patients treated with intravenous natalizumab in the Netherlands were combined for this study. JCV seroconversion was assessed during periods of high (≥15 µg/mL) and low (<15 µg/mL) natalizumab trough concentrations. Low trough concentrations were mainly the result of trough concentration guided personalised extended interval dosing (EID). The seroconversion rates during high and low trough concentrations were compared using a generalised linear mixed model with a Poisson link function. Results A total of 357 patients from 21 hospitals in the Netherlands were included. The annual seroconversion rate of 8.4% observed in patients during periods of high trough concentrations (n=226) was 2.32 times higher than the seroconversion rate of 4.8% in patients during periods of low trough concentrations (n=252) (95% CI=1.32 to 4.08, p=0.0035). Conclusions The seroconversion rate observed in patients with MS with low trough concentrations was substantially lower compared with those with high trough concentrations during natalizumab treatment. This emphasises the importance of personalised EID, where intervals between infusions are prolonged to achieve lower natalizumab trough concentrations, to increase drug safety.

Immunoglobulin G (IgG) is a glycoprotein harboring conserved fragment crystallizable domain glycans, but it can also express variable domain glycans (VDGs). Elevated levels of VDGs are a hallmark of the autoantibodies most specific to rheumatoid arthritis and are also associated with other autoimmune diseases. Nonetheless, the effect of VDGs on IgG function remains poorly understood. This study investigates the impact of VDGs on the ability of antibodies to activate the complement system, a key immune effector mechanism. We demonstrate that VDGs on IgG inhibit the initiation of the classical complement pathway, as evidenced by complement activation assays with IgG displaying varying degrees of VDGs. Structure-function analyses show that VDGs reduce classical complement activation by impeding IgG oligomer formation on antigenic surfaces. As IgG oligomerization is essential for effective C1q binding, VDG-mediated interference with this process lowers IgG's complement activation potential. This impaired ability to recruit complement was further substantiated through direct visualization of IgG oligomer-C1q complexes on antigen-coated vesicles by cryoelectron tomography. Together, these data reveal that VDGs increase the functional diversity of IgG and identify a novel regulatory mechanism that modulates the ability of antibodies to activate one of their main immune effector mechanisms.

Waldenström Macroglobulinemia (WM) is a rare B cell malignancy defined by greater than 10% infiltration of lymphoplasmacytic cells in the bone marrow (BM) and a circulating monoclonal Immunoglobulin M (IgM), while its precursor state IgM monoclonal gammopathy of undetermined significance (MGUS) has <10% BM infiltration. WM and IgM MGUS are unique amongst malignant lymphomas because symptoms and treatment indication may be caused by monoclonal IgM and not by the malignant cell infiltration. These symptoms correlate poorly with IgM levels, suggesting there may be specific biochemical properties of those pathological IgMs, yet IgM structure in IgM gammopathies has not been systematically studied. In healthy individuals, IgM circulates as a pentameric molecule that consists of five covalently linked monomers (H2L2 pairs), a joining (J-) chain and one CD5-Like (CD5L) molecule. In order to gain insight into structural variation of IgM in monoclonal IgM gammopathies, we developed and tested several assays to determine J-chain and CD5L content and polymerization state of IgM from 29 IgM MGUS and WM patients. In multiple cases, IgM was found to be (partially) devoid of J-chain, which associated with differential assembly of IgM into variably sized polymers. Moreover, we found that IgM exceeding ~5 g/L was no longer saturated with CD5L. Relative binding of polymeric Ig receptor varied by over 30-fold. Combined, in this pilot study we demonstrate that structural and functional variation in IgM of IgM MGUS and WM is common. These aberrations in IgM structure may relate to variations in clinical phenotype in IgM monoclonal gammopathies.

OBJECTIVES:The presence of immunoglobulin A (IgA) autoantibodies has been described in many autoimmune diseases, and some of its characteristics, such as IgA dimerization, are considered a sign of a mucosal origin. However, limited information is available about the (patho)physiological conditions leading to the development of monomeric vs dimeric (autoreactive) IgA in humans. Therefore, we investigated IgA dimerization in rheumatic autoimmune diseases with a possible mucosal origin, as well as after vaccination. METHODS:Plasma of patients with rheumatic disease (rheumatoid arthritis [RA], systemic sclerosis [SSc], anti-neutrophil cytoplasmic antibody associated vasculitis [AAV], systemic lupus erythematosus [SLE]), SARS-CoV-2 vaccinated healthy individuals, and healthy controls was used for size exclusion chromatography (SEC). Enzyme-linked immunosorbent assays were performed on SEC fractions to determine the size of IgA. Results were confirmed using western blot and tandem mass spectrometry. RESULTS:The proportion of dimeric IgA was increased for autoantibodies compared to total IgA. This was most evident in RA, AAV, and SLE (dimeric autoreactive IgA ≈ 60%-80% vs total IgA ≈ 20%, SLE ≈ 60% total IgA), but not in SSc. Intramuscular vaccination against SARS-CoV-2 also led to an increased proportion of dimeric anti-spike IgA shortly after vaccination, irrespective of previous mucosal exposure. CONCLUSIONS:These findings indicate that dimeric (autoreactive) IgA responses are associated with newly emerging antigen-specific immune activation, where production temporarily shifts to dimeric IgA. Mucosal triggering is not necessarily always involved in these IgA immune responses. These findings provide key insights into the circumstances for IgA dimerization and suggest that dimeric IgA could serve as a marker for immunological disease activity in autoimmunity.

Antibodies are extensively used in treating various diseases, with over 100 canonical monoclonal antibodies (mAbs) approved. Population pharmacokinetic (PK) models are typically developed for each individual mAb, despite their similarities in size, shape, and susceptibility to lysosomal degradation. However, sparse datasets with limited PK information pose challenges in deriving accurate parameter estimates. Here, we provide a comprehensive overview of 160 published models of 69 mAbs, administered either intravenously or subcutaneously, examining their structural, statistical, and covariate components. Median estimates for the base parameters are linear clearance (0.22 L/d), central volume (3.42 L), peripheral volume (2.68 L), intercompartmental clearance (0.54 L/d), absorption rate (0.25 L/d), and bioavailability (69%). Using these to simulate a ‘generic’ mAb results in plausible kinetics with a terminal half-life of 21 ds. We demonstrated that the median linear clearance was 26% lower in models that included nonlinear target-mediated kinetics, when compared to linear models (0.18 vs. 0.25 L/d). For chimeric mAbs median linear clearance was 50% higher compared to fully human and humanized mAbs. Variability in PK parameter estimates across models was comparable to the inter-individual variability, which have consistently shown to be large for mAbs PK (e.g. 55% vs. 43% for clearance and 25% vs. 30% for central volume, respectively). Our meta-analysis suggests that a priori parameter estimates derived from the large body of existing pharmacokinetic models for mAbs are representative for many mAbs and can facilitate the design of new and/or more complex pharmacokinetic models or assist in dose optimization models.

OBJECTIVES:A substantial proportion of patients with rheumatoid arthritis (RA) discontinue adalimumab due to ineffectiveness. The next treatment step can be another tumour necrosis factor inhibitor (TNFi) or a biological/targeted synthetic disease-modifying antirheumatic drug (b/tsDMARD). Therapeutic drug monitoring (TDM) of serum drug levels may help guide this choice. This study evaluated whether switching treatments based on adalimumab trough levels is more effective than random switching. METHODS:In this 24-week, multicentre, triple-blinded, randomised controlled trial, patients with RA who stopped adalimumab due to inefficacy (disease activity score based on 28 joint count and C-reactive protein [DAS28-CRP] >2.9) were enrolled. Participants were randomly assigned (1:1) to a TDM-based or random switching (control) strategy. The primary outcome was the difference in mean time-weighted DAS28-CRP between groups at 24 weeks. RESULTS:From July 2020 to November 2023, 83 consecutive patients with RA were included, with 78 initiating a new b/tsDMARD (TDM-based group: n = 38; control: n = 40). The mean time-weighted DAS28-CRP was 3.15 in both groups (TDM: SD, 0.99; control: SD, 1.01; 95% CI of difference: -0.46 to 0.47). There were no significant differences between the groups in flare rates, escape medication use, disease activity, or adverse events. Receiver operating characteristic analyses in the control group found no predictive value of adalimumab levels for response to TNFi or non-TNFi. CONCLUSIONS:Switching treatments based on adalimumab trough levels was not more effective than random switching in patients with RA who failed adalimumab treatment. Therefore, serum drug level measurements to guide therapy choices in this context is not recommended.