HYDRASHIFT 2/4 daratumumab, daratumumab Control

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM A. 510(k) Number: K172195 B. Purpose for Submission: New Device C. Measurand: Monoclonal Immunoglobulins (IgG, IgA, IgM) and light chains (kappa, lambda) in serum D. Type of Test: Qualitative E. Applicant: Sebia, Inc. F. Proprietary and Established Names: Hydrashift daratumumab Serum Immunofixation G. Regulatory Information: 1. Regulation section: 21 CFR § 866.5510 Immunoglobulins (A, G, M, D, E) Immunological Test System 21 CFR § 862.1660 Quality Control Material (assayed and unassayed) 2. Classification: Class II 3. Product code: CFF – Immunoelectrophoretic, Immunoglobulins (G, A, M) JJY – Multi-analyte controls, all kinds (assayed) 4. Panel: Immunology (82) Clinical Chemistry (75) H. Intended Uses: 1. Assay: The HYDRASHIFT 2/4 daratumumab test is intended for the qualitative detection of monoclonal proteins in human serum by immunofixation electrophoresis. The kits are to be used in conjunction with the HYDRAGEL IF kits and the semi-automated HYDRASYS 2 electrophoresis apparatus. The proteins, separated by electrophoresis on {1} alkaline buffered agarose gels, are incubated with individual antisera that are specific against gamma (Ig G), alpha (Ig A) and mu (Ig M) heavy chains, and kappa (free and bound) and lambda (free and bound) light chains, respectively. After removing the nonreacted proteins, the immunoprecipitates are stained with acid violet. The electrophoregrams are evaluated visually for the presence of specific reactions with the suspect monoclonal proteins. The HYDRASHIFT 2/4 daratumumab kits remove the daratumumab Ig G, Kappa interference and enable the visual evaluation of the presence or absence of monoclonal proteins on the HYDRAGEL IF kits in patients who have received daratumumab therapy. For In Vitro Diagnostic Prescription Use Only. **Control:** The daratumumab Control is designed for the qualitative quality control of the HYDRASHIFT daratumumab immunofixation procedure performed using the HYDRASYS 2 instrument. The daratumumab Control is designed for laboratory use. It should be used like a human serum sample. For In Vitro Diagnostic Prescription Use Only. 2. **Indications for use:** Same as Intended Uses 3. **Special conditions for use statement:** For prescription use only. 4. **Special instrument requirements:** HYDRASYS 2 electrophoresis apparatus. **I. Device Description:** **Acronyms used in this Decision Memorandum:** SM – Standard Mask DM – Dynamac mask; The SM and DM are used for antisera application CR – Complete Response sCR – Stringent complete response VGPR – Very good partial response Hydragel 2/4 – a configuration of two gels or four gels ELP control – Electrophoresis control IF – Immunofixation Assay kit components: | Item | PN 4639 (20 TESTS) | PN 4640 (40 TESTS) | | --- | --- | --- | | Anti-daratumumab mouse antiserum (ready to use) | 1 vial, 0.4 mL | 1 vial, 0.85 mL | | Sample diluent (ready to use) | 1 vial, 2.2 mL | 1 vial, 2.2 mL | | Green applicators | 1 pack of 10 | 2 packs of 10 | {2} (ready to use) | (15 teeth) | (15 teeth) Reagents required but not supplied: | Item | SEBIA Product Number | | --- | --- | | Daratumumab Control | 4765 | | HYDRAGEL 2 or 4 IF Acid violet - Dynamic mask | 4302, 4304 or 4381* | | Antisera and Fixative for immunofixation IF - Dynamic mask | 4315 | | or | | | HYDRAGEL 2 or 4 IF Acid violet - Standard mask | 4802, 4804 or 4881* | | Antisera and Fixative for immunofixation IF - Standard mask | 4815 | | and | | | Destaining Solution | 4540 | | Hydrasys Wash Solution | 4541 | | HYDRAGEL IF Sample Diluent | 4588 | | Fluidil | 4587 | | DTT Diluent (IF / IT) | 4589 | | Dithiothreitol (DTT) | Not supplied by SEBIA | | Beta-Mercaptoethanol (BME or 2-Mercaptoethanol) | Not supplied by SEBIA | * HYDRAGEL 4 IF MAXI-KIT Equipment and accessories required but not supplied: 1. HYDRASYS 2 System SEBIA: HYDRASYS 2 SCAN PN 1200, HYDRASYS 2 PN 1201, HYDRASYS 2 SCAN FOCUSING PN 1202 or HYDRASYS 2 FOCUSING PN 1203. 2. HYDRASHIFT 2/4 Accessories, SEBIA, PN 1251. It contains: one applicator carrier specific for the HYDRASHIFT daratumumab procedure and 2 guides for anti-daratumumab antiserum application on 15 teeth green applicator. 3. Wet Storage Chamber, PN 1270, supplied with HYDRASYS 2. 4. Container Kit supplied with HYDRASYS 2. 5. Template guide Bar SEBIA supplied with HYDRASYS 2. 6. Accessory Kit for HYDRASYS IF, SEBIA, PN 1260, or Dynamic mask, SEBIA, PN 1255. 7. Pipettes: 10 µL, 20 µL, 100 µL and 200 µL. J. Substantial Equivalence Information: 1. Predicate device name(s): K960669, HYDRAGEL IF, 6 IF, 12 IF PENTA KITS/HYDRAGEL IF, DOUBLE IF, 2 IF, & 4 IF KITS {3} 2. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device K172195 | Predicate K960669 | | Intended Use | The HYDRASHIFT 2/4 daratumumab test is intended for the qualitative detection of monoclonal proteins in human serum by immunofixation electrophoresis. The kits are to be used in conjunction with the HYDRAGEL IF kits and the semi-automated HYDRASYS 2 electrophoresis apparatus. The proteins, separated by electrophoresis on alkaline buffered agarose gels, are incubated with individual antisera that are specific against gamma (Ig G), alpha (Ig A) and mu (Ig M) heavy chains, and kappa (free and bound) and lambda (free and bound) light chains, respectively. After removing the non-reacted proteins, the immunoprecipitates are stained with acid violet. The electrophoregrams are evaluated visually for the presence of specific reactions with the suspect monoclonal proteins. The HYDRASHIFT 2/4 daratumumab kits remove the daratumumab Ig G, Kappa interference and enable the visual evaluation of the presence or absence of monoclonal proteins on the HYDRAGEL IF kits in patients who have received daratumumab therapy. For In Vitro Diagnostic Prescription Use Only. | The HYDRAGEL 1 IF, 2 IF, 4 IF and 9 IF kits are designed for detection of monoclonal proteins in human serum and urine by immunofixation electrophoresis. The kits are used in conjunction with the semi-automated HYDRASYS electrophoresis apparatus. The proteins, separated by electrophoresis on alkaline buffered agarose gels, are incubated with individual antisera that are specific against gamma (Ig G), alpha (Ig A) and mu (Ig M) heavy chains, and kappa (free and bound) and lambda (free and bound) light chains, respectively. After removing the non-reacted proteins, the immunoprecipitates are stained either with acid violet or amidoblack. The electrophoregrams are evaluated visually for the presence of specific reactions with the suspect monoclonal proteins. | | Assay Principle | Same | Agarose Gel Electrophoresis | | Reagents • Gel kit • Antisera Kit | Same Same | HYDRAGEL IF Antisera and Fixative for immunofixation IF | | Antisera Specificity | Same | gamma (IgG), alpha (IgA) and mu (IgM) heavy chains, and kappa (free and bound) and lambda (free and bound) light chains | | Visualization | Same | Acid Violet Gel Staining | 4 {4} | Similarities | | | | --- | --- | --- | | Item | Device K172195 | Predicate K960669 | | of target protein | | | | Results | Same | Qualitative | | Instrument | Same | HYDRASYS electrophoresis apparatus | | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Sample type | Serum | Serum and Urine | | Reagents | Using anti-daratumumab antibody | No anti-daratumumab antibody | | Daratumumab band | Removed from gamma zone into alpha zone | Remains in gamma zone | | Lowest Detectable daratumumab Limit | 0.3 g/L | N/A | # K. Standard/Guidance Document Referenced): CLSI EP7-A2 Interference testing in Clinical Chemistry; Approved Guideline - Second Edition # L. Test Principle: Abnormal bands in serum protein electrophoregrams (SPEP), primarily those in the beta globulin and gamma globulin zones, are suspected to be monoclonal proteins (M-proteins, paraproteins, monoclonal immunoglobulins). The presence of monoclonal protein as indicated by SPEP leads to testing of sample with an IF technique to detect and identify the monoclonal protein components, using the HYDRAGEL IF assay. Daratumumab is a human therapeutic IgG Kappa monoclonal antibody and as such, during the clinical monitoring of patients treated with daratumumab, this antibody is visualized as a band detected by serum protein electrophoresis and immunofixation in the gamma region. The presence of daratumumab can be interpreted as the presence of an endogenous IgG Kappa monoclonal protein, and confound clinical interpretation of the results of the assay. The HYDRASHIFT daratumumab immunofixation procedure performed on HYDRAGEL IF 2/4 gel is based on the creation of a daratumumab/anti-daratumumab antibody complex that is shifted outside the gammaglobulin zone. With the HYDRASHIFT daratumumab procedure, the daratumumab/anti-daratumumab antibody complex is visualized in alpha-1 zone on IgG and Kappa immunofixation tracks and the daratumumab interference is removed from the gamma zone. This can allow for the visualization of any endogenous IgG-kappa monoclonal proreins by IF that may have been masked by the endogenous daratumumab. Daratumumab CONTROL is a qualitative quality control for the assay. {5} 6 # M. Performance Characteristics: ## 1. Analytical performance: All results met the manufacturer’s pre-determined acceptance criteria. ### a. Precision/Reproducibility: To evaluate precision (repeatability and reproducibility), a panel of ten serum samples, including one normal serum sample (daratumumab control) one electrophoresis (ELP) control and eight serum samples with different monoclonal protein components, were tested using the HYDRASHIFT 2/4 daratumumab procedure used in conjunction with each of the following kits: HYDRAGEL 4 IF Acid violet Standard mask, and HYDRAGEL 4 IF Acid violet Dynamic mask. The samples tested for precision are described in the table below: | Sample Number | Sample Type | | --- | --- | | 1 | Normal (Daratumumab control) | | 2 | IT/IF control (ELP) IgG, L + IgA, K + IgM, L | | 3 | IgG, K | | 4 | IgG, L | | 5 | IgA, K | | 6 | IgA, L | | 7 | IgM, K | | 8 | IgM, L | | 9 | Kappa free | | 10 | Lambda free | ### Within-run precision, repeatability: Each of the above 10 samples was tested four times within the same gel. Samples 1 and 2 were run without addition of daratumumab as controls. Samples 3–10 were run with daratumumab (1g/L) and without daratumumab. All samples were run with both Standard Mask (SM) and Dynamic Mask (DM) for a total of eight runs per sample. The gels results were read and evaluated by two readers for the presence or absence of monoclonal proteins and the presence of the daratumumab/anti-daratumumab antibody complex visualized in the alpha-1 zone on IgG and Kappa immunofixation tracks. Repeatability was evaluated visually for the concordance of band presence from the same sample. Results showed 100% of concordance for the same sample, and between the same sample tested with SM and DM and between readers. The results are summarized below: - Native samples were 100% concordant when run on SM and DM - Replicates of daratumumab spiked samples were 100% concordant between SM and DM. Samples with monoclonal proteins other than IgG Kappa were also 100% concordant with native samples - As expected, results that were different between native and spiked samples showed shifted bands in the IgG and Kappa immunofixation tracks from the {6} gammaglobulin zone and the band was visualized in the alpha-1 zone *Reproducibility between days, between lots and between instruments:* Reproducibility was tested with the same panel of 10 samples, as described above. Samples 1 and 2 were run without addition of daratumumab as controls. Samples 3-10 were tested with and without daratumumab (1g/L). The samples were run with the four gel configuration on SM and DM using three HYDRASYS 2 instruments, with three lots of HYDRASHIFT IF kits, over three days resulting in nine replicates per sample on SM and nine replicates per sample on DM. All results were read by two readers. Reproducibility was evaluated visually for the concordance of monoclonal proteins and the daratumumab/anti-daratumumab antibody complex. Results that were different between native and spiked sample showed, as expected the shifted band of the daratumumab/anti-daratumumab antibody complex in the alpha-1 zone of IgG and Kappa Immunofixation tracks. There was 100% concordance for the all the samples between days, instruments and lots, between SM and DM application methods and between the two readers. An additional study was done to demonstrate reproducibility between two and four gel configuration and between DM and SM applicators. 163 patient samples were used that included negative samples and samples with monoclonal protein components including IgG-Kappa. The samples included 80 daratumumab-treated samples and 62 samples that were tested native and spiked with daratumumab. Assays were performed with HYDRASHIFT 2/4 daratumumab procedure used in conjunction with: HYDRAGEL 4 IF Acid Violet Standard mask kit HYDRAGEL 4 IF Acid Violet Dynamic mask kit HYDRAGEL 2 IF Acid Violet Standard mask kit HYDRAGEL 2 IF Acid Violet Dynamic mask kit There was 100% concordance between the two and four gel configuration and between the DM and SM procedures. *b. Linearity/assay reportable range:* Not applicable *c. Traceability, Stability, Expected values (controls, calibrators, or methods):* Traceability: The daratumumab control and the anti-daratumumab antibody are manufactured by Sebia and are traceable to internal reference controls. Daratumumab Control: The daratumumab control is a qualitative control with a visual interpretation. It is manufactured from human sera spiked with daratumumab. It is traceable to an internal reference control sample. Sebia recommends to run an assayed control serum CONTROL SEBIA PN 4765 after each change of lot of a reagent. {7} # Stability: The test reagents except for the daratumumab control and the anti-daratumumab antibody were cleared in the predicate device. All the components are stable until the expiration date indicated on the kit box or on the components labels. Detection Antibody: the anti-daratumumab detection antibody is murine anti-human daratumumab IgG. The antibody stability was tested in an accelerated stability study for two months at $18 - 22^{\circ}\mathrm{C}$ with three lots of anti-daratumumab antibody. One month accelerated stability is equal to one year stability at $2 - 8^{\circ}\mathrm{C}$ . Results were compared to results of anti-daratumumab stored at $2 - 8^{\circ}\mathrm{C}$ for two months, using four gel configuration and SM and DM application methods, with four serum samples with monoclonal proteins (IgM Kappa, IgA Kappa and two IgG Kappa). Open vial stability was tested in real time at $2 - 8^{\circ}\mathrm{C}$ . The results are summarized in the table below. Additional real-time stability testing is ongoing. Sample stability: Real-time stability was evaluated on six IgG Kappa samples from patients treated with daratumumab. Testing was performed using SM and the two gel configuration. Results were compared to the same sample at time zero. There was $100\%$ concordance between reference (T0) and test results. Testing was performed for: Three months at $-26^{\circ}\mathrm{C}$ to $-30^{\circ}\mathrm{C}$ For 10 days at $2 - 8^{\circ}\mathrm{C}$ For three days at $19 - 25^{\circ}\mathrm{C}$ Control Stability: The daratumumab control stability was tested with three lots using DM and SM with the four gel configuration for using a normal sample tested in four replicates. Accelerated stability study was performed for three months at $18 - 22^{\circ}\mathrm{C}$ . One month accelerated stability is equal to one year stability at $2 - 8^{\circ}\mathrm{C}$ . Results were compared to the same lots tested at time zero and at the same time points when stored at $2 - 8^{\circ}\mathrm{C}$ . Real time stability was performed to evaluate open vial stability at $2 - 8^{\circ}\mathrm{C}$ and at $-26$ to $-30^{\circ}\mathrm{C}$ . In addition the control was tested as reconstituted, frozen, and then subjected to freeze/thaw cycles. The results are summarized in the table below. Real-time stability is ongoing. The stability claims of the HYDRASHIFT assay is summarized in the table below: | | Shelf life | Open vial | | --- | --- | --- | | IgG Anti-daratumumab | 2 years at 2–8°C | 6 months at 2–8°C | | Daratumumab treated samples | | 1 week 2–8°C 3 days at 19–25°C 3 months at -26 to -30 °C | | Daratumumab control | 3 years at 2–8°C | 1 week at 2–8°C 6 months at -26 / - 30°C 20 cycles (frozen and thawed) | {8} 9 HYDRASHIFT 2/4 Kit 2 years at 2-8°C d. Detection limit: Sensitivity: A study was done to determine the threshold detection value of visualization of the daratumumab and/or the daratumumab/anti-daratumumab antibody complex. Six serum samples, including two normal serum samples and four serum samples with different monoclonal components, were spiked with daratumumab at different concentrations for a final daratumumab concentration of 3.0, 2.0, 1.5, 1.0, 0.5, 0.3, 0.2, 0.1 and 0.0 g/L. Samples were analyzed with the HYDRASHIFT 2/4 daratumumab procedure using the 4 gel configuration and SM and DM application method. The serum samples that were used in the study are described in the table below: | Sample | Sample type | Total Band Protein Concentration | | --- | --- | --- | | 1 | Pool of normal serum | 73.3 g/L | | 2 | Pool of normal serum | 37.3 g/L | | 3 | IgG Kappa | 5.0 g/L | | 4 | IgA Kappa | 8.3 g/L and 1.7 | | 5 | IgM Kappa | 2.9 g/L | | 6 | IgM Lambda and Kappa free | 1.2 g/L | The results showed that the limit of detection for visualization of the daratumumab/anti-daratumumab antibody complex at the alpha-1 zone of G Kappa tracks is 0.3 g/L. e. Analytical specificity: Interference: The performance of the HYDRASHIFT 2/4 daratumumab procedure was evaluated in the presence of common interfering factors. Testing was performed based CLSI guideline EP7-A2. No interference with the HYDRASHIFT 2/4 daratumumab procedure, was detected with the intereferents up to the concentrations listed in the table below: {9} | Endogenous Interfering substance | Concentration | | --- | --- | | Bilirubin | 20 mg/dL (342 μM) | | riglycerides | 3,00 g/dL (34.5 mM) | | Hemoglobin | 2 g/L | | Rheumatoid factor | 2000 UI/mL | | Human Anti-mouse Antibody HAMA | Titer: 640 | | Drugs | Concentration | | Pomalidomide | 1 mg/L | | Lenalidomide | 4 mg/L | | Dexamethasone | 1 mg/L | | Bortezomib | 2 mg/L | A separate study was done with 42 samples to demonstrate that treatment of the sample with dithiothreitol (DTT) does not interfere with the HYDRASHIFT 2/4 daratumumab procedure used in conjunction with the HYDRAGEL 4 IF Acid Violet kit. The results demonstrated that DTT treatment does not affect the performance of the HYDRASHIFT 2/4 daratumumab procedure. f. Assay cut-off: Not applicable ## 2. Comparison studies: a. Method comparison with predicate device: Method comparison was done at two U.S. sites with 198 samples at site 1 and 172 samples at site 2 as detailed in the table below. The study was done using the 4 gel configuration, with both SM and DM, and two readers. For samples with visual bands in the IgG and Kappa tracks, the HYDRASHIFT 2/4 daratumumab demonstrated that shifting of the daratumumab band allowed evaluation of whether endogenous daratumumab is present. The samples tested at the two sites are described in the table below: | Site | Normal samples* | Pathological samples** | IgG Kappa samples | IgG Kappa + other monoclonal components samples | Total samples*** | | --- | --- | --- | --- | --- | --- | | Site 1 | 42 (including 37 treated with daratumumab) | 156 (including 76 treated with daratumumab) | 55 | 14 | 198 | | Site 2 | 38 (including 34 treated with daratumumab) | 134 (including 64 treated with daratumumab) | 50 | 14 | 172 | {10} * Without monoclonal component ** With monoclonal component(s) *** 172/198 samples were analyzed at both sites. Samples were run with HYDRAGEL IF Acid Violet kit procedure (predicate) and compared to samples run with the HYDRASHIFT 2/4 daratumumab procedure (the test method). The results were evaluated as "Same" or as "Different". Not unexpectedly, there were a large number of samples that had a different characterization/results between the two methods (113 samples at Site 1 and 98 samples at Site 2). For 100% of these samples, the difference of interpretation was due to the presence of daratumumab in the IgG Kappa monoclonal component in the sample, interpreted in the predicate as positive. That band was different in the test method because the daratumumab/anti-daratumumab complex was shifted out of the gamma zone of the gel. Therefore, the discordant results are explained by the assay desing differences. There was 100% concordance between the results of the 172 samples tested at the two sites. b. Matrix comparison: Not applicable 2. Clinical studies: a. Clinical Sensitivity: Not applicable b. Clinical specificity: Not applicable Not applicable 4. Clinical cut-off: Not applicable 5. Expected values/Reference range: Not applicable Q. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. R. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision.