Exposure-disease response analysis of natalizumab in subjects with multiple sclerosis.

Natalizumab, a human immunoglobulin monoclonal antibody that targets α4ß1/α4ß7 integrin, is an effective therapy approved for the treatment of multiple sclerosis (MS). The objective of this analysis was to develop a population exposure-response model utilizing gadolinium-enhancing (Gd) lesion count data from four clinical studies and annualized relapse rate (ARR) data from three clinical studies. The natalizumab exposures were derived for the individuals using a population pharmacokinetic model. A log-linear exposure effect on Gd lesion count and ARR adequately characterized the relationship between exposure and disease response. In the case of the Gd lesion count model, a bimodal model that distributed subjects into two subpopulations based on low or high baseline Gd lesion count provided a superior goodness of fit. The mean (95% confidence interval) slopes from the exposure-Gd lesion count model and exposure-ARR model are -0.0903 (-0.100, -0.081) and -0.0222 (-0.026, -0.015) (mg/L)-1, respectively. From these slopes, it can be inferred that both Gd lesion count and ARR decrease with increasing exposure to natalizumab in MS subjects. Model-based simulations demonstrated that although reductions in Gd lesion count and ARR were observed with lower doses (75, 150, or 200 mg), only the dose of 300 mg every 4 weeks (q4w) was associated with an ARR ≤0.25 and was considered clinically effective. The results from the exposure-Gd lesion count and exposure-ARR models thus support the appropriateness of the approved natalizumab dose (300 mg q4w) in MS subjects.

Translational biomarkers: from discovery and development to clinical practice.

The refinement of disease taxonomy utilizing molecular phenotypes has led to significant improvements in the precision of disease diagnosis and customization of treatment options. This has also spurred efforts to identify novel biomarkers to understand the impact of therapeutically altering the underlying molecular network on disease course, and to support decision-making in drug discovery and development. However, gaps in knowledge regarding disease heterogeneity, combined with the inadequacies of surrogate disease model systems, make it challenging to demonstrate the unequivocal association of molecular and physiological biomarkers to disease pathology. This article will discuss the current landscape in biomarker research and highlight strategies being adopted to increase the likelihood of transitioning biomarkers from discovery to medical practice to enable more objective decision making, and to improve health outcome.

Risk of natalizumab-associated progressive multifocal leukoencephalopathy.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is associated with natalizumab treatment. We quantified the risk of PML in patients with multiple sclerosis, according to the presence or absence of three risk factors: positive status with respect to anti-JC virus antibodies, prior use of immunosuppressants, and increasing duration of natalizumab treatment. METHODS: We used data from postmarketing sources, clinical studies, and an independent Swedish registry to estimate the incidence of PML among natalizumab-treated patients with multiple sclerosis, according to positive or negative status with respect to anti-JC virus antibodies, prior or no prior use of immunosuppressants, and duration of treatment (1 to 24 months vs. 25 to 48 months). Blood samples were available for anti-JC virus antibody testing from 5896 patients with multiple sclerosis and from 54 patients with multiple sclerosis who were treated with natalizumab and in whom PML later developed. RESULTS: As of February 29, 2012, there were 212 confirmed cases of PML among 99,571 patients treated with natalizumab (2.1 cases per 1000 patients). All 54 patients with PML for whom samples were available before the diagnosis were positive for anti-JC virus antibodies. When the risk of PML was stratified according to three risk factors, the risk of PML was lowest among the patients who were negative for anti-JC virus antibodies, with the incidence estimated to be 0.09 cases or less per 1000 patients (95% confidence interval [CI], 0 to 0.48). Patients who were positive for anti-JC virus antibodies, had taken immunosuppressants before the initiation of natalizumab therapy, and had received 25 to 48 months of natalizumab treatment had the highest estimated risk (incidence, 11.1 cases per 1000 patients [95% CI, 8.3 to 14.5]). CONCLUSIONS: Positive status with respect to anti-JC virus antibodies, prior use of immunosuppressants, and increased duration of natalizumab treatment, alone or in combination, were associated with distinct levels of PML risk in natalizumab-treated patients with multiple sclerosis. (Funded by Biogen Idec and Elan Pharmaceuticals.).

Anti-JC virus antibody levels in serum or plasma further define risk of natalizumab-associated progressive multifocal leukoencephalopathy.

OBJECTIVE: The increased risk of progressive multifocal leukoencephalopathy (PML) with natalizumab treatment is associated with the presence of anti-JC virus (JCV) antibodies. We analyzed whether anti-JCV antibody levels, measured as index, may further define PML risk in seropositive patients. METHODS: The association between serum or plasma anti-JCV antibody levels and PML risk was examined in anti-JCV antibody-positive multiple sclerosis (MS) patients from natalizumab clinical studies and postmarketing sources. For PML and non-PML patients, the probabilities of having an index below and above a range of anti-JCV antibody index thresholds were calculated using all available data and applied to the PML risk stratification algorithm. Longitudinal stability of anti-JCV antibody index was also evaluated. RESULTS: Anti-JCV antibody index data were available for serum/plasma samples collected >6 months prior to PML diagnosis from 71 natalizumab-treated PML patients and 2,522 non-PML anti-JCV antibody-positive patients. In patients with no prior immunosuppressant use, anti-JCV antibody index distribution was significantly higher in PML patients than in non-PML patients (p < 0.0001). Among patients who were anti-JCV antibody negative at baseline in the AFFIRM and STRATIFY-1 trials, 97% remained consistently negative or below an index threshold of 1.5 over 18 months. Retrospective analyses of pre-PML samples collected longitudinally from PML patients displayed sustained higher anti-JCV antibody index over time. INTERPRETATION: Anti-JCV antibody levels in serum/plasma, measured as index, may differentiate PML risk in anti-JCV antibody-positive MS patients with no prior immunosuppressant use. Continued evaluation of anti-JCV antibody index and PML risk is warranted.

Changes to anti-JCV antibody levels in a Swedish national MS cohort.

BACKGROUND: The anti-JC virus (JCV) antibody status has been introduced to stratify patients with multiple sclerosis (MS) for higher or lower risk of progressive multifocal leukoencephalopathy (PML). OBJECTIVE: To assess the potential utility of anti-JCV antibody levels for earlier diagnosis or prediction of PML. METHODS: An analytically validated antibody assay was used to determine serological status, normalised optical density values, and dilution titres for anti-JCV antibodies. The method was applied to stored sera of 1157 patients with MS including five cases of PML, all enrolled in the Swedish pharmacovigilance study for natalizumab (NAT). Anticytomegalovirus (CMV) and antivaricella-zoster (VZV) antibody levels served as controls. RESULTS: Prior to treatment with NAT, anti-JCV antibody levels were stable in the anti-JCV positive patients. During therapy, a slight decrease in anti-JCV and anti-VZV antibody levels, but not anti-CMV antibody levels, was observed. All five patients who developed PML showed a mild to moderate increase in anti-JCV antibody levels at time of PML diagnosis; pre-PML samples suggested that this increase might start already prior to diagnosis of PML. CONCLUSIONS: Treatment initiation with NAT may lead to a slight decrease in anti-JCV and anti-VZV antibody levels, suggestive of a mild suppressive effect of NAT on antibody levels. Our findings in five cases of PML demonstrate that the onset of PML can be accompanied by increasing anti-JCV antibodies in serum. Monitoring of anti-JCV antibody levels could potentially be used as a tool for prediction or earlier diagnosis of PML during NAT treatment for MS. Further studies are warranted.

The effect of plasma exchange on serum anti-JC virus antibodies.

OBJECTIVE: Natalizumab, a highly effective treatment for multiple sclerosis (MS) and Crohn's disease, is associated with progressive multifocal leukoencephalopathy (PML). Upon suspicion or diagnosis of PML, plasma exchange (PLEX) is performed to remove natalizumab from the circulation, allowing immune reconstitution of the central nervous system. Since PLEX may also remove other circulating antibodies, we examined the effects of PLEX on serum immunoglobulin (IgG) and anti-JC virus (JCV) antibody levels in MS patients with and without PML. METHODS: Serum samples from 12 natalizumab-treated patients without PML collected before, during and after PLEX were tested for IgG isotypes using a commercial assay, and for anti-JCV antibodies using a two-step enzyme-linked immunosorbent assay. Five natalizumab-treated PML patients who underwent PLEX were also tested for anti-JCV antibodies. RESULTS: PLEX produced a two- to three-fold reduction in all IgG isotypes. Among patients without PML, 42% (five of 12 patients) had detectable anti-JCV antibodies before PLEX; in these patients, anti-JCV antibodies were reduced approximately two- to five-fold, with levels returning to 50-100 percent of baseline two weeks after the final PLEX. The five PML patients, all of whom had detectable anti-JCV antibodies before PLEX, experienced similar reductions in anti-JCV antibody levels following PLEX. CONCLUSIONS: Our results indicate that PLEX effectively removes circulating antibodies; however, levels of endogenous anti-JCV antibody, unlike exogenously administered natalizumab, were replenished relatively quickly following PLEX. While interpretation of anti-JCV antibody levels during or within two weeks after PLEX may be problematic, humoral JCV immunity is not abolished by PLEX and antibody levels are rapidly restored.

Anti-JC virus antibody prevalence in a multinational multiple sclerosis cohort.

JC virus (JCV) is an opportunistic virus known to cause progressive multifocal leukoencephalopathy. Anti-JC virus (Anti-JCV) antibody prevalence in a large, geographically diverse, multi-national multiple sclerosis (MS) cohort was compared in a cross-sectional study. Overall, anti-JCV antibody prevalence was 57.6%. Anti-JCV antibody prevalence in MS patients ranged from approximately 47% to 68% across these countries: Norway, 47.4%; Denmark, 52.6%; Israel, 56.6%; France, 57.6%; Italy, 58.3%; Sweden, 59.0%; Germany, 59.1%; Austria, 66.7% and Turkey, 67.7%. Prevalence increased with age (from 49.5% in patients < 30 years of age to 66.5% in patients ≥ 60 years of age; p < 0.0001 comparing all age categories), was lower in females than in males (55.8% versus 61.9%; p < 0.0001) and was not affected by prior immunosuppressant or natalizumab use.

Anti-John Cunnigham virus antibody prevalence in multiple sclerosis patients: baseline results of STRATIFY-1.

OBJECTIVE: A study was undertaken to define the prevalence of anti-JC virus (JCV) antibodies in multiple sclerosis (MS) patients and to evaluate the analytical false-negative rate of a 2-step anti-JC virus antibody assay. METHODS: STRATIFY-1 is an ongoing, longitudinal, observational study of relapsing MS patients in the United States who are being treated or considering treatment with natalizumab. Baseline serum and plasma samples were collected for anti-JC virus antibody detection using an analytically validated, 2-step, virus-like particle-based enzyme-linked immunosorbent assay. Urine was collected for JC virus DNA detection. RESULTS: At baseline (n = 1,096), overall anti-JC virus antibody prevalence was 56.0% (95% confidence interval [CI], 53.0-59.0) in STRATIFY-1 patients, with an assay false-negative rate of 2.7% (95% CI, 0.9-6.2). Prevalence was significantly lower in females (53.4%; 95% CI, 49.9-56.8) than males (64.3%; 95% CI, 58.2-70.0) and increased with age, p = 0.0019 and p = 0.0001, respectively. Prevalence was similar in patients regardless of natalizumab exposure or prior immunosuppressant use, p = 0.9709 and p = 0.6632, respectively. STRATIFY-1 results were generally consistent with those observed in another large North American cohort, TYGRIS-US (n = 1,480). INTERPRETATION: Baseline results from STRATIFY-1 are consistent with other studies utilizing this assay that demonstrate a 50 to 60% prevalence of anti-JC virus antibodies, a low false-negative rate, and an association of increasing age and male gender with increasing anti-JC virus antibody prevalence. Neither natalizumab exposure nor prior immunosuppressant use appear to affect prevalence. Longitudinal data from STRATIFY-1 will confirm the stability of anti-JC virus antibody prevalence over time.

In vivo pharmacology and toxicology evaluation of polyethylene glycol-conjugated interferon beta-1a.

Human interferon (IFN) ß has well established beneficial effects in treating relapsing forms of multiple sclerosis, but current first-line treatment requires frequent (from daily to weekly) parenteral administration. A 20-kDa polyethylene glycol (PEG)-conjugated IFN ß-1a (PEG-IFN ß-1a) is being developed to decrease the frequency of administration and improve patient convenience and compliance. We present pharmacokinetic (PK) and pharmacodynamic (PD) parameters, immunogenicity, and safety of PEG-IFN ß-1a in Rhesus monkeys in support of a phase 1 clinical trial. Two single-dose PK/PD studies and one 5-week repeat-dose toxicity study compliant with good laboratory practice were conducted. The PK of IFN ß-1a and PEG-IFN ß-1a were modeled with a two-compartment model, and the link between drug concentration and neopterin response (PD marker) was described with an indirect stimulatory model. PEG-IFN ß-1a showed greater exposure, longer half-life, lower clearance, and reduced volume of distribution than unmodified IFN ß-1a. Consistent with the pharmacology of type I IFNs, PEG-IFN ß-1a resulted in the elevation of neopterin concentration, a transient body temperature increase, and a reversible lymphocyte count decrease. As expected, neutralizing antibodies to PEG-IFN ß-1a formed in almost all monkeys after 5 weeks of treatment, which resulted in significantly reduced drug exposure and abrogation of neopterin induction. There were no drug-related adverse effects at doses up to 100 µg/kg (11 MIU/kg) given subcutaneously or intramuscularly once weekly for 5 weeks. The no-observed-adverse-effect level was determined to be 100 µg/kg (11 MIU/kg), the highest dose tested.

Anti-JC virus antibodies: implications for PML risk stratification.

OBJECTIVE: A study was undertaken to establish an enzyme-linked immunosorbent assay (ELISA) to detect JC virus (JCV)-specific antibodies in multiple sclerosis (MS) patients, and to evaluate its potential utility for identifying patients at higher or lower risk (ie, risk stratification) of developing progressive multifocal leukoencephalopathy (PML). METHODS: A 2-step assay for detecting and confirming the presence of anti-JCV antibodies in human serum and plasma was developed and demonstrated to be both sensitive and specific. ELISA cutpoints were statistically established using sera from >800 MS patients from natalizumab clinical studies. Subsequently, this assay was used to determine the presence of anti-JCV antibodies in natalizumab-treated PML patients where serum samples were collected 16-180 months prior to the diagnosis of PML. RESULTS: In our evaluation of natalizumab-treated MS patients, 53.6% tested positive for anti-JCV antibodies, with a 95% confidence interval of 49.9 to 57.3%. The false-negative rate of the ELISA was calculated to be approximately 2.5%, with an upper 1-sided confidence limit of 4.4%. Notably, we observed anti-JCV antibodies in all 17 available pre-PML sera samples, which was significantly different from the 53.6% seropositivity observed in the overall MS study population (p < 0.0001). INTERPRETATION: This 2-step assay provides a means to classify MS patients as having detectable or not detectable levels of anti-JCV antibodies. The finding that all 17 of the pre-PML samples that were available tested seropositive, and none tested seronegative, warrants further research on the clinical utility of the anti-JCV antibody assay as a potential tool for stratifying MS patients for higher or lower risk of developing PML.

Increased plasma concentrations of cytoskeletal and Ca2+-binding proteins and their peptides in psoriasis patients.

BACKGROUND: The mechanisms underlying psoriatic pathogenesis are not fully understood and might be elucidated by identifying novel disease-related molecular markers, including autoantigens. METHODS: We used 2 proteomic methods to analyze plasma samples from 20 psoriasis patients and 20 matched healthy donors. The first method focused on evaluating changes in glycoprotein concentrations and the plasma proteome, and the second method assessed endogenous proteolytic activity by analyzing the low molecular weight component of plasma. RESULTS: The integrated proteomic and peptidomic analysis identified a number of proteins and their fragments present at different concentrations in the plasma of psoriasis patients and healthy donors. We used ELISA to independently verify the changes in the concentrations of several of these proteins. One intriguing finding, increased concentrations of cytoskeletal and actin-binding proteins and their peptides in psoriatic plasma, suggested disease-related cell leakage of these proteins and their increased proteolysis. Among the increased proteins and peptides were thymosin beta 4, talin 1, actin gamma, filamin, and profilin. Increased concentrations of Ca(2+)-binding proteins calgranulins A and B in psoriatic plasma were also observed, confirming previous reports, and appeared to be relevant to the increase of cytoskeletal components. Another notable change in psoriatic plasma was a striking decrease in fibrinogen fragments. CONCLUSIONS: The identified increased concentrations of cytoskeletal proteins, their peptides, and calgranulins in psoriatic plasma, as well as the underlying altered protease activity, are proposed to be related to psoriasis pathogenesis.

Population Pharmacokinetics and Target Engagement of Natalizumab in Patients With Multiple Sclerosis.

Natalizumab (humanized immunoglobulin G4 antibody targeting alpha-4 integrins) is a highly efficacious treatment for relapsing-remitting multiple sclerosis (RRMS) that has been in clinical use since 2006. However, natalizumab pharmacokinetic (PK) characteristics and concentration alpha-4 integrin saturation relationships have not been well described in the scientific literature. Therefore, clinical data from 11 studies were pooled and analyzed to characterize the PK and pharmacodynamic (PD) properties of natalizumab in RRMS subjects. Natalizumab PK was best described using a 2-compartment model with linear first-order and Michaelis-Menten elimination. Subcutaneous absorption of natalizumab was characterized using first-order absorption with lag time. The relationship between natalizumab concentration and alpha-4 integrin saturation (PD) was best described by a direct response model with a sigmoidal effect on alpha-4 integrin saturation mediated by a maximum effect relationship with natalizumab concentrations. Covariate analysis showed that body weight, product formulations, and the presence of antinatalizumab antibodies were the main covariates affecting natalizumab PK, whereas age and formulations affected PD. The use of simulations based on a pharmacokinetic-pharmacodynamic model showed that covariates, although statistically significant, are not expected to have any clinical impact at the approved clinical dosing regimen of natalizumab (300 mg once every 4 weeks).

Efficacy, safety, and pharmacokinetics of natalizumab in Japanese multiple sclerosis patients: A double-blind, randomized controlled trial and open-label pharmacokinetic study.

BACKGROUND: Natalizumab, an anti-α4 integrin monoclonal antibody, has demonstrated efficacy in phase 2 and 3 studies of predominantly Caucasian patients with relapsing-remitting multiple sclerosis (RRMS). OBJECTIVE: To evaluate the efficacy, safety, pharmacokinetics (PK), and pharmacodynamics (PD) of natalizumab in Japanese RRMS patients. METHODS: This multicenter, phase 2 study included an open-label PK/PD study in 12 patients (part A) and a double-blind, placebo-controlled, randomized (computer-generated sequence) study in 94 patients (part B). For part B, patients received intravenous natalizumab 300mg (n=47) or placebo (n=47) every 4 weeks. The primary efficacy endpoint was the rate of development of new active lesions (gadolinium-enhancing or new/enlarging T2 lesions) over 24 weeks. Clinical relapses and safety were also assessed. RESULTS: New active lesions developed at a significantly lower mean rate in natalizumab-treated patients (0.06 lesions/24 weeks) than in placebo-treated patients (0.35 lesions/24 weeks) (p<0.001). The annualized relapse rate was 0.53 for natalizumab and 1.73 for placebo (p<0.001). Twice as many natalizumab-treated patients (79%) as placebo-treated patients (38%) were relapse-free (p<0.001). The safety, PK, and PD profiles of natalizumab in this study were consistent with data in Caucasian RRMS patients. CONCLUSIONS: In Japanese RRMS patients, natalizumab treatment every 4 weeks for 24 weeks was well tolerated and reduced the development of new brain lesions and relapses (Funded by Biogen; ClinicalTrials.gov identifier: NCT01440101).

Reversibility of the effects of natalizumab on peripheral immune cell dynamics in MS patients.

OBJECTIVE: To characterize the reversibility of natalizumab-mediated changes in pharmacokinetics/pharmacodynamics in patients with multiple sclerosis (MS) following therapy interruption. METHODS: Pharmacokinetic/pharmacodynamic data were collected in the Safety and Efficacy of Natalizumab in the Treatment of Multiple Sclerosis (AFFIRM) (every 12 weeks for 116 weeks) and Randomized Treatment Interruption of Natalizumab (RESTORE) (every 4 weeks for 28 weeks) studies. Serum natalizumab and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured using immunoassays. Lymphocyte subsets, α4-integrin expression/saturation, and vascular cell adhesion molecule-1 (VCAM-1) binding were assessed using flow cytometry. RESULTS: Blood lymphocyte counts (cells/L) in natalizumab-treated patients increased from 2.1 × 109 to 3.5 × 109. Starting 8 weeks post last natalizumab dose, lymphocyte counts became significantly lower in patients interrupting treatment than in those continuing treatment (3.1 × 109 vs 3.5 × 109; p = 0.031), plateauing at prenatalizumab levels from week 16 onward. All measured cell subpopulation, α4-integrin expression/saturation, and sVCAM changes demonstrated similar reversibility. Lymphocyte counts remained within the normal range. Ex vivo VCAM-1 binding to lymphocytes increased until ≈16 weeks after the last natalizumab dose, then plateaued, suggesting reversibility of immune cell functionality. The temporal appearance of gadolinium-enhancing lesions was consistent with pharmacodynamic marker reversal. CONCLUSIONS: Natalizumab's effects on peripheral immune cells and pharmacodynamic markers were reversible, with changes starting 8 weeks post last natalizumab dose; levels returned to those observed/expected in untreated patients ≈16 weeks post last dose. This reversibility differentiates natalizumab from MS treatments that require longer reconstitution times. Characterization of the time course of natalizumab's biological effects may help clinicians make treatment sequencing decisions. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that the pharmacodynamic markers of natalizumab are reversed ≈16 weeks after stopping natalizumab.

Incidence, characterization, and clinical impact analysis of peginterferon beta1a immunogenicity in patients with multiple sclerosis in the ADVANCE trial.

BACKGROUND: Efficacy of interferon beta in multiple sclerosis (MS) can be dampened in patients who develop neutralizing antidrug antibodies (NAbs). Peginterferon beta1a is an interferon conjugated with a polyethylene glycol (PEG) moiety. Pegylation increases a drug's half life and exposure, and may also reduce immunogenicity. OBJECTIVE: The objective of this study was to characterize the incidence and impact of immunogenicity to peginterferon beta1a over 2 years in patients with MS. METHODS: Patients with relapsing-remitting MS (N = 1512) were randomized to subcutaneous peginterferon beta1a 125 µg every 2 or 4 weeks, or placebo, for 1 year; patients in the placebo group were rerandomized to active treatment in year 2. The incidence and titers of binding antibodies (BAbs) and NAbs to interferon and antibodies to PEG (anti-PEG) were assessed in analytically validated assays. The clinical impact of immunogenicity on relapse and magnetic resonance imaging endpoints was evaluated. RESULTS: Over 2 years, 6%, less than 1%, and 7% of patients developed anti-interferon BAbs, NAbs, and anti-PEG antibodies, respectively. There was no discernible clinically meaningful effect of antibody status on the pharmacodynamic, efficacy, or safety parameters evaluated, although these analyses were limited by the low incidence of treatment-emergent antibodies. CONCLUSION: The treatment effect of peginterferon beta1a in patients with relapsing-remitting MS is not expected to be attenuated by immunogenicity.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Incurred sample reproducibility and stability assessment in a cell-based drug concentration assay.

Joleen White is Principal Scientist in Translational Sciences at Biogen Idec. Throughout her career, she has applied her background in biophysical protein chemistry to pharmaceutical development in therapeutic indications with significant unmet medical need. In her current role, she supports method development and regulated bioanalysis of biomarkers, biopharmaceuticals, and immunogenicity in biological samples from nonclinical and clinical studies. Her experience with measuring macromolecules includes enzymes, monoclonal antibodies, Fc fusions, oligonucleotides, PEGylated proteins, and other novel protein constructs. She has supported studies from discovery through all phases of development including GLP nonclinical, clinical, and post-marketing commitments. Incurred samplereproducibility is one aspect of in-study validation, with white papers outlining expectations for chromatographic assays and immunoassays. This manuscript outlines an approach for performing incurred sample reproducibility for a bioequivalence study using a cell-based assay, with the complication of time elapsed between original and repeat assays. The incurred sample reproducibility passed the pre-established acceptance criteria of 45% for at least 2/3 of the samples: 174/216 samples (80.6%). Data trends between the two crossover arms were qualitatively similar. The passed incurred sample reproducibility and stability further supports the validity of the original study conclusion that the two manufacturing processes were bioequivalent. This illustrates one approach to extrapolating industry and regulatory recommendations for situations outside current guidance.