Summary

• Hydrashift 2/4 Daratumumab Immunofixation Assay
  • Kirchhoff et al (2021)¹ reported results from a single-center retrospective review of clinical history and laboratory findings of patients with multiple myeloma (MM) that evaluated the impact of the Hydrashift assay. In cohort 1a, there were39 timepoints from 8 patients whose M-protein co-migrated with daratumumab on serum protein electrophoresis (SPEP) and IFE. The Hydrashift assay allowed correct interpretation both at timepoints (n=16) where both an M-protein and daratumumab were visible and timepoints (n=23) where only daratumumab was visible by the Hydrashift assay. The Hydrashift assay thus allowed correct interpretation of all cases of co-migration of patients’ M-protein and daratumumab.
  • Thoren et al (2019)² reported results from a study that evaluated the performance of the Hydrashift assay in patients with MM who had received DARZALEX-based therapy. In 27 cases, an M-protein and extra immunoglobulin G kappa (IgGκ) band were distinguished by IFE alone. In 26/27 cases, the Hydrashift assay confirmed that the IgGκ band was daratumumab. In 11 cases, the patient’s IgGκ M-protein comigrated with daratumumab. In these cases, the Hydrashift assay showed that the disease band was present in these 11 patients.
  • Tang et al (2018)³ reported results from a retrospective study that evaluated the frequency and pattern of detection of therapeutic-monoclonal antibodies (tmAbs) by SPEP and IFE in patients with relapsed/refractory multiple myeloma (RRMM) who received DARZALEX or elotuzumab. T-mAb was distinguishable from M-protein in 16/26 courses, with daratumumab detected in 9/9 (100%). During induction therapy (weekly infusions), daratumumab was consistently detected; however, it was infrequently detected in patients receiving biweekly or monthly (maintenance) infusions. Daratumumab disappeared from follow-up SPEP/IFE in 4/6 patients receiving maintenance DARZALEX. In 2 patients whose DARZALEX course ended during the study period, t-mAb was detected by SPEP/IFE up to 11 days after treatment cessation.
  • Irimia et al (2016)⁴ reported results from a study that compared the Hydrashift 2/4 daratumumab assay with laboratory-developed Daratumumab-Specific Immunofixation Electrophoresis Reflex Assay (DIRA) test for the displacement of daratumumab on IFE. Concordance was 100% for the 51 samples tested with laboratory developed DIRA test and Hydrashift 2/4 daratumumab assay (28 negative DIRA, 14 positive DIRA, and 9 doubtful DIRA). For 48 samples tested at diagnosis, the anti-daratumumab antibody (Ab) shifted daratumumab with no effect on the patients’ Mspike with 100% specificity.
• SPIFE Anti-daratumumab Reagent
  • Baloda et al (2022)⁵ reported results from a pilot study that evaluated the ability of a novel derivatized anti-daratumumab Ab to eliminate DARZALEX’s interference with SPEP and IFE. In 14 samples from patients receiving DARZALEX, SPEP and IFE revealed complete disappearance of the cathodal restricted population after SPIFE anti-daratumumab pretreatment. For 6 samples from patients without documented DARZALEX, the monoclonal populations persisted despite SPIFE anti-daratumumab treatment.
• DIRA
  • McCudden et al (2016)⁶ reported results from a study that described the validation of DIRA in distinguishing daratumumab from endogenous M-protein while assessing its limit of sensitivity, specificity, and reproducibility. DIRA was developed using a mouse anti-daratumumab Ab to differentiate between endogenous myeloma protein and daratumumab. DIRA sensitivity by IFE at 0.1 vs 0.2 g/L DARZALEX concentration in normal human samples (NHS) and MM serum samples, respectively, was 80% vs 100% and 90% vs 100%, respectively. DIRA sensitivity by SPEP at 0.2 g/L DARZALEX concentration in NHS was 100%, and at 0.1 vs 0.2 g/L DARZALEX concentration in MM serum samples, it was 30% vs 100%, respectively.
  • Caillon et al (2015)⁷ reported results from a study that assessed the biochemical response in patients with MM who received DARZALEX by evaluating the relevant biochemical assays using DIRA with SPEP/IFE based on isotype at diagnosis, electrophoresis mobility, and residual M-spike. At 0.1 g/dL concentration of antidaratumumab, the complex between daratumumab and anti-daratumumab completely shifted to the low-globulin zone. When the residual spike is about 10% of the initial one (around very good partial response [VGPR]), to shift daratumumab, DIRA-SPE is used for patients with a daratumumab comigrated M-protein. When the residual spike is <0.1 g/dL, DIRA-IFE is used for patients with a daratumumab comigrated M-protein to ensure that malignant M-protein is no longer detectable.
• Free Light Chain (FLC) Assay
  • Rosenberg et al (2016)⁸ reported results from a study that evaluated the interference of DARZALEX on the FLC assay. In 10 normal samples spiked with DARZALEX, a monoclonal band was identified on IFE in the gamma (γ)zone in patients who received DARZALEX. In 20 samples with known IgGκ Mprotein, no significant effect of DARZALEX on measured kappa (κ-), lambda (λ-) or the FLC ratio was observed.
• Clinical Assay
  • Axel et al (2014)⁹ reported results from a study that developed a clinical assay to mitigate DARZALEX interference with SPEP and serum IFE, and clinical response assessment. Incubation of DARZALEX with an anti-daratumumab Ab in phosphatebuffered saline (PBS) or NHS, prior to serum IFE, shifted daratumumab migration in IFE gels. At 1:2 ratio of daratumumab:anti-idiotype Abs, a complete daratumumab shift was observed. In serum samples taken from patients with MM, the anti-daratumumab Ab altered the daratumumab bands without changing the endogenous M-protein migration pattern. Labeling the anti-idiotype Ab with biotin or Alexa-fluor tags altered the daratumumab complex, providing more separation between the daratumumab signal and endogenous M-protein.
• Up-Conversion Fluorescence Lateral-Flow Immunoassay (LFA)
  • Liu et al (2024)¹⁰ reported results from a study that determined the interference of DARZALEX in SPEP samples via up-conversion fluorescence LFA. The limit of detection (LOD) for daratumumab was identified at a signal level of 33.9, correlating to a daratumumab concentration of 0.020 mg/mL. The dynamic range of the LFA strip was determined to be 0.037-3.0 mg/mL, using a coefficient of variance (CV) of 20% as the threshold for imprecision. Analytical interference testing revealed no signal above the LOD in normal serum samples (n=3) and hemolytic whole blood samples (n=2, hemolysis triggered by diluting the samples in phosphate-buffer saline with Tween 20 and bovine serum albumin [PBST-B]). No significant signal interference was observed in a serum sample containing adalimumab at 3.0 mg/mL and a sample containing an FLC-polyclonal antibody (FLC-pAb) at 3.0 mg/mL. The LFA strip detected daratumumab or isatuximab in 21 samples, consistent with patient records showing 20 patients on daratumumab and one on isatuximab.
• Mass Spectrometry-Based Assay
  • Eveillard et al (2021)¹¹ reported results from a study that determined the concordance between Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the routine SPEP/IFE/FLC strategy, assessed the performance of MALDI-TOF-MS in differentiating IgGκ monoclonal proteins from daratumumab, and characterized the advantages and limitations in the serial analysis of up to 10 samples per patient (clinicaltrials.gov identifier: NCT03290950). Using IFE as the reference, MALDI-TOF-MS was 85.4% concordant in 170/199 samples (P<0.001). Using the routine serum panel as reference, MALDI-TOF-MS was 84.9% concordant for 169/199 samples (P<0.001). Among patients who obtained CR (N=9), MALDI-TOF-MS detected a monoclonal protein in 22/48 (46%) post-CR samples up to the end of treatment (EOT; range, cycle 2 to EOT) in 6 patients.

PRODUCT LABELING

• DARZALEX (daratumumab) [Prescribing Information]. Horsham, PA: Janssen Biotech, Inc.; www.janssenlabels.com/package-insert/product-monograph/prescribing-information/DARZALEX-pi.pdf
• DARZALEX FASPRO (daratumumab and hyaluronidase-fihj) [Prescribing Information]. Horsham, PA: Janssen Biotech, Inc.; www.janssenlabels.com/package-insert/product-monograph/prescribing-information/DARZALEX+Faspro-pi.pdf

BACKGROUND

• DARZALEX may be detected on SPEP and IFE assays used for monitoring disease monoclonal immunoglobulins (M-protein). This can lead to false-positive SPEP and IFE assay results for patients with IgGκ myeloma protein impacting initial assessment of CRs by International Myeloma Working Group (IMWG) criteria. In patients with persistent VGPR, where DARZALEX interference is suspected, consider using a validated DARZALEX-specific IFE assay to distinguish daratumumab from any remaining endogenous M-protein in the patient’s serum, to facilitate determination of a CR.^12,13
• In January 2018, Sebia received 510(k) clearance from the United States (US) Food and Drug Administration (FDA) for its Hydrashift 2/4 daratumumab assay, intended to be used with Sebia’s Hydragel IFE test, for the qualitative detection of M-proteins in human serum by IFE.¹⁴ The Sebia Hydrashift 2/4 daratumumab in vitro diagnostic (IVD) can be used to assess response in DARZALEX-treated patients with IgGκ MM.¹⁵

clinical Data

Hydrashift 2/4 Daratumumab Immunofixation Assay

Impact of Hydrashift 2/4 Daratumumab Immunofixation Assay

Kirchhoff et al (2021)¹ reported results from a single-center retrospective review of clinical history and laboratory findings of patients with MM that evaluated the impact of the Hydrashift assay.

Study Design/Methods

• Clinical history and laboratory results of patients were retrospectively reviewed.
• Patients included were as follows:
  • Cohort 1: All Hydrashift results and corresponding SPEP/IFE results obtained from July 2018 to March 2020 (n=280 samples from 96 unique patients)
  • Cohort 1a: Patients whose M-protein comigrated with daratumumab on SPEP and IFE and who had serial Hydrashift results (n=39 samples from 8 unique patients)
  • Cohort 2: Patients whose M-protein peak was different from that of daratumumab on SPEP (n=38 samples from 38 unique patients)
• SPEP was performed by capillary electrophoresis using the Capi-3 Protein€ 6 kit (Sebia) on the Capillarys 3 Tera analyzer (Sebia).
• IFE was performed using the Hydrasys 2 analyzer (Sebia) using either the Hydragel 9 or Hydragel 4 IFE kit (Sebia).
• The Hydrashift assay was performed on the Hydrasys 2 analyzer using the Hydrashift 2/4 daratumumab kit (Sebia) and Hydragel 4 IFE kit.

Results

• In cohort 1, among 48/96 patients, the original clone was IgGκ.
  • In 43 patients, M-proteins comigrated with daratumumab on IFE, and 5 patients had Mproteins that were distinct from daratumumab.
• In cohort 1a, there were 16 timepoints where both the M-protein and daratumumab were visible by the Hydrashift assay.
  • The peak on SPEP ranged from 0.08 to 0.17 g/dL in these samples (mean, 0.11 g/dL).
  • There were 23 timepoints where only daratumumab was visible by the Hydrashift assay.
    • The Hydrashift assay allowed correct interpretation of these 23 false-positive IFE results.
  • The daratumumab peak ranged from 0.06 to 0.14 g/dL (mean, 0.09 g/dL).
• In cohort 2, the concentration of daratumumab ranged from 0.02 to 0.20 g/dL (mean, 0.11 g/dL).
  • The daratumumab peak is typically visible in the first SPEP after the initiation of DARZALEX therapy (first SPEP performed 6-43 days [mean, 15 days] after starting therapy; received a minimum of 2 doses before their routine SPEP and IFE).
• Of the 280 samples, 91 resulted in a gel showing only the daratumumab complex, 148 resulted in a gel showing both the patient's M-protein and daratumumab complex, 39 resulted in a gel showing only the patient’s M-protein, and 2 showed neither the patient’s M-protein nor the daratumumab complex.

Performance of Hydrashift 2/4 Daratumumab Immunofixation Assay

Thoren et al (2019)² reported results from a study that evaluated the performance of the Hydrashift assay in patients with MM who had received DARZALEX-based therapy.

Study Design/Methods

• Waste clinical samples from patients with MM (N=40) who had received DARZALEXbased therapy were collected and analyzed using the Hydrashift assay and a standard IFE gel for comparison.
  • Both assays were performed on the Hydrasys 2 system (Sebia) using the Hydragel 4 IFE kit (Sebia).

Results

• In samples with sufficient DARZALEX concentration, shift of the daratumumab-related band was most evident and distinct from the M-protein band on IFE.
• At 27/40 instances, the patient’s M-protein and an additional IgGκ band were distinguishable by IFE alone.
  • When the M-protein was an isotype other than IgGκ (n=20) or if the M-protein was IgGκ (n=5) or free κ (n=2) with a different migration pattern than daratumumab, the bands were distinguished by IFE alone.
  • In 26/27 cases, the Hydrashift assay confirmed the additional band of daratumumab.
• In 13/40 instances, daratumumab was indistinguishable from the patient’s disease by IFE alone, as only a single IgGκ band was visible, and these patients had a history of IgGκ or free κ myeloma.
  • In 11/13 patients, pretreatment SPEP and IFE results showing migration of the original clone was available, proving that their immunoglobulin G (IgGκ) M-protein comigrated with daratumumab.
    • According to baseline SPEP and IFE data, the Hydrashift assay showed that the disease band was present in these 11 patients.
• For 2 patients, one with IgGκ myeloma and the other with κ light chain myeloma, pretreatment IFE findings were unavailable.
• The Hydrashift assay showed that the IgGκ band observed on IFE was only because of daratumumab and that the patients’ disease was not detectable.

Detection of tmAbs Using the Hydrashift 2/4 Daratumumab Immunofixation Assay

Tang et al (2018)³ reported results from a retrospective study that evaluated the frequency and pattern of detection of t-mAbs by SPEP and IFE in patients with RRMM who received DARZALEX or elotuzumab.

Study Design/Methods

• A pharmacy chart review was performed over a 19-month period for patients with ≥1 subsequent SPEP/IFE available for evaluation (N=22; DARZALEX, 14 courses; elotuzumab, 12 courses).
• SPEP analysis was done using the Sebia Capillarys system (Sebia USA, Norcross, GA) and IFE using the Sebia Hydrasys (Sebia USA).

Results

• Over the 19-month study period, the number of infusions per patient ranged from 2 to 41, and 8 patients were continuing therapy at the end of the study period.
• The first follow-up SPEP or IFE was between 7 and 49 days after treatment initiation.
• The earliest t-mAb detection was 7 days after the start of therapy (MM1), with latest detection at 101 days (MM2).
  • T-mAb was detected on the first follow-up SPEP/IFE in 80% of patients having detectable tmAb.
• In 3 DARZALEX cases, wherein the M-protein level was at least 0.9 g/dL throughout the therapy, t-mAb shared same migration and isotype with the patient’s myeloma Mprotein.
• The presence of t-mAb could not be definitively assessed in 10/26 occurrences of therapy, either because of t-mAb having the same migration and isotype as the significant persistent myeloma M-protein or because of the lack of follow-up IFE testing.
• T-mAb could be distinguished from the myeloma M-protein in 16 courses of therapy with informative follow-up testing owing to its distinct migration or isotype.
  • In these patients, daratumumab was detected in 9/9 courses, where it was present at a concentration of ≤0.1 g/dL.
• During induction therapy (weekly infusions), daratumumab was consistently detected; however, it was infrequently detected in patients receiving biweekly or monthly (maintenance) infusions.
• Daratumumab disappeared from follow-up SPEP/IFE in 4/6 patients receiving maintenance DARZALEX.
• In 2 patients whose DARZALEX course ended during the study period, t-mAb was detected by SPEP/IFE up to 11 days after treatment cessation.

Comparison of Hydrashift 2/4 Daratumumab Assay with Laboratory-developed DIRA test

Irimia et al (2016)⁴reported results from a study that compared the Hydrashift 2/4 daratumumab assay with laboratory-developed DIRA test for the displacement of daratumumab on IFE.

Study Design/Methods

• The Hydrashift 2/4 daratumumab assay, developed by Sebia, used the antidaratumumab Ab produced by Janssen, which was modified to allow the migration of the daratumumab/anti-daratumumab complexes toward the α-globulin fraction on IFE.⁴
• Ninety-nine samples from ongoing DARZALEX clinical trials were assessed for analytical performances, such as sensitivity, specificity, and comparability with the original DIRA test.⁴

Results

• IFE was positive at diagnosis for serum samples from 309/324 (95.4%) patients.¹⁶
• In 119/309 (38.5%) cases, the M-spike partially or totally comigrated with daratumumab detected in the serum.¹⁶
• Anti-daratumumab displacement assay was required in 66/309 (21.4%) cases of IgGκMM or κ-light chain multiple myeloma (LCMM), where standard IFE could not distinguish daratumumab from endogenous M-spike.¹⁶
• Concordance was 100% for the 51 samples tested with laboratory developed DIRA test and Hydrashift 2/4 daratumumab assay (28 negative DIRA, 14 positive DIRA, and 9 doubtful DIRA).⁴
• Daratumumab/anti-daratumumab complexes were detected in the α-globulin fraction with a sensitivity of 200 mg/L.^4,16
  • Daratumumab/anti-daratumumab complex was difficult to visualize when DARZALEX concentrations were <200 mg/L, but daratumumab was completely removed from the γ-globulin fraction with no trace left for all concentrations tested.
• For 48 samples tested at diagnosis, the anti-daratumumab Ab shifted daratumumab with no effect on the patients’ M-spike with 100% specificity.^4,16

SPIFE Anti-daratumumab Reagent

Elimination of DARZALEX Interference Using the SPIFE Anti-daratumumab Reagent

Baloda et al (2022)⁵ reported results from a pilot study that evaluated the ability of a novel derivatized anti-daratumumab Ab to eliminate DARZALEX’s interference with SPEP and IFE.

Study Design/Methods

• A characteristic daratumumab electrophoretic pattern (a cathodal monoclonal IgGκ pattern) was identified in 20 samples in the clinical laboratory from May to June 2017, wherein 14/20 patients had documented DARZALEX infusions.
• Serum samples were collected in serum separator tubes; SPEP and IFE were run as part of the patient’s standard care, and further testing was performed with remnant specimens to detect the SPIFE anti-daratumumab Ab.
  • The SPIFE anti-daratumumab reagent is an anti-daratumumab polyclonal Ab that creates daratumumab/SPIFE anti-daratumumab complexes; this causes the daratumumab to disappear into the polyclonal background.
• All 20 samples were tested to determine if SPIFE anti-daratumumab could specifically and effectively deplete daratumumab.
• To test the effect of dilution factor on the quantification of the bands on SPEP, the neat samples with samples diluted with saline were compared with those diluted with SPIFE anti-daratumumab Ab.

Results

• In the 14 samples from patients receiving DARZALEX, SPEP and IFE revealed complete disappearance of the cathodal restricted population after SPIFE anti-daratumumab pretreatment.
• For the 6 samples from patients without documented DARZALEX infusion, the monoclonal populations persisted despite SPIFE anti-daratumumab treatment, and their M-protein represented disease-associated monoclonal antibody (mAb) rather than administered t-mAbs, indicating the specificity of SPIFE anti-daratumumab for daratumumab over disease M-protein.
• The differences between the original sample M-protein quantification of disease‑associated M-protein and the diluted samples (saline and anti-daratumumab diluted) ranged from 1% to 22% (0.1-2.4 g/L dilution of serum by saline and antidaratumumab), with a mean absolute value of 10.18% (0.8 g/L dilution of serum by saline and anti-daratumumab; n=11), when assessed for the percentage M-protein (percentage of area under the curve) and absolute values.

DIRA

Validation of DIRA

McCudden et al (2016)⁶ reported results from a study that described the validation of DIRA in distinguishing daratumumab from endogenous M-protein while assessing its limit of sensitivity, specificity, and reproducibility. DIRA was developed using a mouse antidaratumumab Ab to differentiate between endogenous myeloma protein and daratumumab.

Study Design/Methods

• Human serum samples from patients with MM or healthy donors were collected from either a commercial source (n=51) or DARZALEX-treated patients (n=33) from clinical trials.
  • Clinical trials where DARZALEX was being used as a monotherapy (GEN501 and SIRIUS) or as combination therapy with lenalidomide (ongoing study, GEN503; ClinicalTrials.gov Identifier: NCT01615029) were considered.
• To evaluate whether anti-daratumumab bound to and shifted the migration pattern of daratumumab, the mouse anti-daratumumab antibody was spiked into DARZALEXcontaining serum and resolved by IFE/SPEP.
  • IFE was performed on semi-automatic Hydrasys or Hydrasys 2 using Maxikit Hydragel 4IFE or 9IFE (Sebia, Norcross, GA, USA).
  • SPEP was performed on Capillarys using the Capillarys Protein 6 kit (both from Sebia).
• Patients were used for validation and DIRA testing if they had low-level (<5 g/L) or negative SPEP, and repeated positive IgGκ-IFE results owing to potential DARZALEX interference.
• For DIRA, anti-daratumumab or saline was spiked into baseline or DARZALEX-treated patient serum using a standardized method to measure the following:
  • Migration of control daratumumab with anti-daratumumab
  • Lack of migration of the baseline M-protein with the addition of anti-daratumumab
  • Shift in the migration pattern of the putative daratumumab band relative to the DARZALEX control in DARZALEX-treated serum samples
  • Presence vs absence of a nondaratumumab M-protein band (that denoted a DIRApositive vs DIRA-negative result)

Results

• The sensitivity of DIRA for DARZALEX was at ~0.2 g/L. The sensitivity of DIRA by IFE and SPEP in NHS and MM serum samples spiked with different concentrations of DARZALEX is present in Table: DIRA Sensitivity at Different DARZALEX Concentration in NHS and MM Serum Samples by IFE and SPEP.

• In commercial samples spiked with 0.5 or 1 g/L DARZALEX, there was a 100% shift in anti-daratumumab Ab at both concentrations.
• No shift in the M-protein occurred with the addition of anti-daratumumab alone in any of the samples.
• In 8% (4/51) of samples, a faint band appeared with the addition of anti-idiotype at both concentrations with IgG antisera.
• In 10/10 (100%) DARZALEX-treated patient samples, results were consistent across all 3 independent experiments.
• There was 100% concordance when 2 independent reviewers assessed daratumumab and M-protein levels across multiple experiments based on predefined acceptance criteria.

Biochemical Response Assessment by DIRA

Caillon et al (2015)⁷ reported results from a study that assessed the biochemical response in patients with MM who received DARZALEX by evaluating the relevant biochemical assays using DIRA with SPEP/IFE based on isotype at diagnosis, electrophoresis mobility, and residual M-spike.

Study Design/Methods

• DARZALEX was added to normal serum to produce interference.
• Multiple concentrations of DARZALEX were tested to identify appropriate concentration of anti-daratumumab.
• DIRA-SPEP and DIRA-IFE were performed for patients with LCMM and IgGκ MM, both receiving DARZALEX.

Results

• At 0.1 g/dL concentration of anti-daratumumab, the complex between daratumumab and anti-daratumumab completely shifted to the low-globulin zone.
• For patients with LCMM with no monoclonal component detected at diagnosis or those with an M-protein that has a different electrophoretic migration from daratumumab, DIRASPEP is enough to ensure that emergence of a new M-spike is really due to DARZALEX.
• When the residual spike is about 10% of the initial one (around VGPR), to shift daratumumab, DIRA-SPE is used for patients with a daratumumab comigrated Mprotein.
• When the residual spike is <0.1 g/dL, DIRA-IFE is used for patients with a daratumumab comigrated M-protein to ensure that malignant M-protein is no longer detectable.

FLC Assay

Detection of DARZALEX interference on the FLC Assay

Rosenberg et al (2016)⁸ reported results from a study that evaluated the interference of DARZALEX on the FLC assay.

Study Design/Methods

• Thirty serum samples from patients with known IgGκ (with or without DARZALEX, n=20) and non-IgGκ M-proteins (n=10) were spiked with DARZALEX at a final concentration of 1.0 mg/mL, followed by FLC analysis.
• In further 20 samples (10 non-IgGκ with M-proteins mentioned above and 10 without Mproteins) with or without DARZALEX, IFE was performed to determine the presence and migration of daratumumab.

Results

• In the 10 normal samples spiked with DARZALEX, a monoclonal band was identified on IFE in the γ-zone in patients who received DARZALEX.
• In the 20 samples with known IgGκ M-protein, no significant effect of DARZALEX on measured κ-, λ- or the FLC ratio was observed.
  • Levels of free κ, λ-light chains and FLC ratio in the presence and absence of DARZALEX are presented in Table: Levels of Free κ, λ-Light Chains and FLC Ratio in the Presence and Absence of DARZALEX.
• DARZALEX at a concentration of 2 mg/mL (n=5) alone without serum gave values ranging from 0.4 to 1.4 mg/L for free κ at the lower LOD of the assay, indicating that even at peak serum concentrations, daratumumab is unlikely to interact with the FLC assay.

Clinical Assay

Mitigation of DARZALEX Interference Using a Clinical Assay

Axel et al (2014)⁹ reported results from a study that developed a clinical assay to mitigate DARZALEX interference with SPEP and serum IFE, and clinical response assessment.

Study Design/Methods

• Serum samples from normal human donors and patients with MM were obtained commercially.
• SPEP was done by separating the serum samples on agarose gel.
• Serum IFE assays were performed using the Helena Quickgel Kit (Helena Laboratories, Beaumont, TX).
  • Antisera against γ (IgG), α (immunoglobulin A [IgA]), and mu (μ; immunoglobulin M [IgM]) heavy chains and free and bound κ- and λlight chains were used to characterize the monoclonal component of each sample.
• Daratumumab was detected in PBS and serum samples by spiking DARZALEX into PBS (preincubated with mouse anti-daratumumab) or NHS (incubated with 1000 μg/mL daratumumab and 2000 μg/mL anti-daratumumab Ab) at varying concentrations (5000, 2500, 750, 500, 250 μg/mL).
• IFE was used to detect IgG and κ in each sample for validating the daratumumab reflex assay.

Results

• IFE detected DARZALEX concentration as low as 250 μg/mL, which was below the steady-state level of 1000 μg/mL normally observed in patients treated with DARZALEX.
• In 50% of serum samples of patients with MM, the M-protein overlapped with daratumumab, making it difficult to differentiate the daratumumab signal from the endogenous M-protein, which further lead to poor monitoring of the disease progression.
• Incubation of daratumumab with an anti-daratumumab Ab shifted its migration in SPEP gels, depending on the concentration of the anti-daratumumab Ab.
• Incubation of daratumumab with an anti-daratumumab Ab in PBS or NHS, prior to serum IFE, shifted daratumumab migration in IFE gels.
• At 1:2 ratio of daratumumab:anti-idiotype Abs, a complete daratumumab shift was observed.
• In serum samples taken from patients with MM, the anti-daratumumab Ab altered the daratumumab bands without changing the endogenous M-protein migration pattern.
• Additionally, labeling the anti-idiotype Ab with biotin or Alexa-fluor tags altered the daratumumab complex, providing more separation between the daratumumab signal and endogenous M-protein.

Up-Conversion Fluorescence LFA

Evaluation of the Interference of DARZALEX in SPEP Samples via Up-Conversion Fluorescence LFA

Liu et al (2024)¹⁰ reported results from a study that determined the interference of daratumumab in SPEP samples via up-conversion fluorescence LFA.

Study Design/Methods

• His-tagged cluster of differentiation (CD38) was procured from Sino Biological, Wayne, PA.
• Daratumumab biosimilar and adalimumab biosimilar were obtained from Ushelf, Hopkinton, MA.
• Anti–FLC-pAb was purchased from Agilent Technologies, Santa Clara, CA.
• Drug-free serum (Lyphochek 456) was sourced from Bio-Rad Laboratories, Hercules, CA.
• A total of 43 serum samples were collected from Stanford Health Care under institutional review board protocol 66075. The sample cohort included 20 samples with a suspected daratumumab interference and 23 samples with a positive abnormal band from endogenous M-protein.
• All SPEP tests were performed using a HYDRAGEL 15 kit on a HYDRASYS 2 gel electrophoresis instrument (Sebia, Evry, France).

Up-Conversion Fluorescence LFA Strip:

• An up-conversion fluorescence LFA strip was designed for the semi-quantitative detection of daratumumab in clinical samples.
• The LFA strip consisted of a sample pad, conjugation pad, nitrocellulose (NC) membrane, and wicking pad.
• The conjugation pad, made of glass fiber, was prepared by dispensing up-conversion nanoparticle–conjugated-CD38, was dried at 37C, and stored at room termperature.
• A mouse anti-human IgG antibody (Bioeast Biotech, Zhejiang, China) at a concentration of 1.0 mg/mL was dispensed onto the test line of the NC membrane, and daratumumab was dispensed onto the control line.
• The LFA strip components were assembled on a plastic adhesive backing card, with a 2 mm overlap for fluid migration.
• The strip was cut into 4 mm width using a strip cutter and placed into a strip cassette for testing.
• The test involved applying a diluted serum sample to the LFA strip, with subsequent fluorescence quantification using a customized up-conversion fluorescence reader.

Assay Evaluation:

• The up-conversion fluorescence LFA strip was evaluated for its LOD, dynamic range, and analytical interference.
• The LOD was determined using a series of diluted daratumumab standards, with calculations based on the 95% confidence interval.
• Daratumumab was spiked in drug-free serum to prepare a 3.0 mg/mL standard sample, which was further diluted by a dilution factor of 3, at concentrations of 1.0, 0.33, 0.11, 0.037, 0.012, and 0.0041 mg/mL, respectively.
• The dynamic range was established with a CV set at 20% (n=4).
• Daratumumab concentration in serum samples was calculated by interpolating the sample readout into a 4-parameter logistic curve fitted to the standard data.
• Analytical interference was assessed with normal serum, hemolytic whole blood samples, and daratumumab samples spiked with other antibodies.

Results

Assay Performance:

• The LOD for daratumumab was identified at a signal level of 33.9, correlating to a daratumumab concentration of 0.020 mg/mL.
• The dynamic range of the LFA strip was determined to be 0.037-3.0 mg/mL, using a CV of 20% as the threshold for imprecision.
• Analytical interference testing revealed no signal above the LOD in normal serum samples (n=3) and hemolytic whole blood samples (n=2, hemolysis triggered by diluting the samples in PBST-B), indicating resistance to sample matrix background in up-conversion fluorescence detection.
• No signal over the threshold was observed in a serum sample containing adalimumab at 3.0 mg/mL and a sample containing FLC-pAb at 3.0 mg/mL, confirming the analytical specificity of the LFA strip.
• Further testing with daratumumab and adalimumab biosimilar (1.0 mg/mL) in drug-free serum showed insignificant interference when daratumumab concentration was above 0.11 mg/mL. However, at or below 0.037 mg/mL, daratumumab readouts were suppressed but remained above the LOD.

Correlation With Patient Medical Records:

• The LFA strip was tested on 43 serum samples from patients with SPEP-positive results. Among these, 20 samples were suspected of daratumumab interference.
• The LFA strip detected daratumumab or isatuximab in 21 samples, consistent with patient records showing 20 patients on daratumumab and 1 patient on isatuximab.
• Concordance was observed in 40 samples between LFA strip results and SPEP observations, with 19 positive and 21 negative matches; 3 samples were discordant.
• Three discordant results were noted:
  • Sample S5 had a daratumumab concentration of 0.065 mg/mL, which was within the LFA strip's dynamic range but below the visible daratumumab band level.
  • Sample S7 tested positive for isatuximab, aligning with patient records but not with suspected daratumumab interference.
  • Sample S35, suspected of daratumumab interference, tested negative on the LFA strip, likely due to high monoclonal FLC κ levels rather than mAb therapy.
• Isatuximab also appeared as a monoclonal band in SPEP gels, complicating differentiation from daratumumab interference. Detection of isatuximab in isatuximab-containing serum samples may serve as an additional utility of the LFA strip.

Mass Spectrometry-Based Assay

Evaluation of Concordance Between MALDI-TOF-MS and the SPEP/IFE/FLC Strategy

Eveillard et al (2021)¹¹ reported results from a study that determined the concordance between MALDI-TOF-MS and the routine SPEP/IFE/FLC strategy, assessed the performance of MALDI-TOF-MS in differentiating IgGκ monoclonal proteins from daratumumab, and characterized the advantages and limitations in the serial analysis of up to 10 samples per patient (clinicaltrials.gov identifier: NCT03290950).

Study Design/Methods

• Each patient had serum drawn at 10 timepoints: baseline, day 15 of cycle 1, the first day of each cycle from cycle 2 to cycle 8, and at the EOT.
• SPEP, IFE, and FLC measurements were performed at all timepoints except for day 15 of cycle 1.
  • SPEP was performed using capillary electrophoresis (Capillarys 3, Sebia Inc., Norcross, GA).
  • IFE was performed on the Hydrasys 2 instrument using the Hydragel 9 IFE kit (Sebia Inc.).
  • FLC assay was performed on the Optilite automated analyzer using the Freelite™ reagent (The Binding Site, Birmingham, UK).
• Immunoglobulins were purified from all available serum samples collected over time for 23 patients (number of samples=222) using magnetic beads coated with polyclonal sheep Abs specific for human IgG or IgA heavy chains or κ or λ light chains, following which they were analyzed using MALDI-TOF-MS.

Results

• Twenty-three patients with newly diagnosed multiple myeloma (NDMM) who were treated with 8 cycles of quadruplet DARZALEX-based combination therapy (carfilzomiblenalidomide-dexamethasone-DARZALEX) were included.
• Results at day 15 of cycle 1 were as follows:
  • Daratumumab was visible in the spectra for 18/23 patients.
  • A separate peak could not be detected for daratumumab in 5/23 patients because the IgGκ monoclonal protein concentration was high and close to the mass of daratumumab at this timepoint.
  • Five patients had a mass to charge ratio (m/z) value within 30 units of DARZALEX.
• At cycle 2, 2/5 patients had an overlap between daratumumab and the monoclonal protein.
• At cycle 3 and later timepoints, daratumumab was visible in all samples because the monoclonal protein concentration decreased, resulting in the detection of separate peaks for daratumumab and the monoclonal protein.
• Using IFE as the reference, MALDI-TOF-MS was concordant in 170/199 samples (85.4%, P<0.001).
  • Matrix-assisted laser desorption ionization (MALDI) was positive for 158 (79%) samples, and IFE was positive for 143 (72%) samples.
• Using the routine serum panel as reference, MALDI-TOF-MS was concordant for 169/199 samples (84.9%, P<0.001).
  • Both MALDI and the panel were positive for 158 samples (79%).
• Concordance of MALDI-TOF-MS, IFE, and routine serum panel is presented in Table: Concordance of MALDI-TOF-MS, IFE, and Routine Serum Panel (SPEP, IFE, and FLC) (n=199).
• Among patients who obtained CR (N=9), MALDI-TOF-MS detected a monoclonal protein in 22/48 (46%) post-CR samples up to the EOT (range, cycle 2 to EOT) in 6 patients.
  • Performance of MALDI-TOF-MS in differentiating IgGκ monoclonal proteins from daratumumab at different timepoints is presented in Table: M-Protein Detection Rates at Different Timepoints.

LITERATURE SEARCH

A literature search of MEDLINE^®, Embase^®, BIOSIS Previews^®, and Derwent Drug File (and/or other resources, including internal/external databases) was conducted on 16 August 2024.

For streamlining purposes, systemic reviews, review articles, and case reports have been excluded.