Stability of nivolumab in its original vials after opening and handing in normal saline bag for intravenous infusion.

OBJECTIVES: Nivolumab is an anti-programmed cell death-1 monoclonal antibody, approved for numerous indications in oncohaematological cancers. It is available as solution for infusion at 10 mg/ml. In accordance with the Summary of Product Characteristics (SmPCs), the product is stable for 24 h at 2-8 °C after dilution. However, to anticipate the needs and constraints related to the handling of the product, the aim was to obtain additional information that will contribute to the risk assessment in case of deviation. Potential changes in the stability of Opdivo® leftovers (10 mg/ml) and diluted nivolumab in normal saline solution (2 mg/ml) over a period exceeding 24 h, at different temperatures and after freezing/thawing cycles were studied. METHODS: Turbidimetry, Ultraviolet (UV)-spectroscopy, dynamic light scattering and chromatography were used to evaluate physicochemical stability. Potential pharmacological variations were monitored in vitro by a functional binding inhibition method. RESULTS: No change was detected after 1 month of storage at 2-8 °C neither after 7 days at 40 °C. Although slight changes were detected only after 3 months under 2-8 °C, major changes were found for the same period of time at 40 °C (variants in the distribution profile, slight increase in oligomers and fragments and UV spectral modifications). Physical instability was observed upon 3 freeze/thaw cycles, with the appearance of a new protein population associated with an increase in polydispersity index. CONCLUSION: In conclusion, our results provide additional rationale to the SmPCs, regarding the use of leftovers, reassignment of bags, pre-preparation or breaking the cold chain for Nivolumab.

A measuring method for occupancy of immune checkpoint inhibitors in the cell surface.

Monoclonal antibodies, including immune-checkpoint inhibitors, are becoming popular in treatments of many cancers and connective tissue diseases. However, little is known about how long the antibodies combine with antigens on targeted cells or how this duration of binding associates with therapeutic efficacy or potential adverse events. Here, we show the principle and the results of a feasible method for measuring the antibodies' occupancy on the targeted cells using two different detecting antibodies in conjunction with different fluorochromes. Nivolumab occupancy was measured using two detecting antibodies, MIH4 and EH12.2, which are commercially available in vitro (programmed cell death-1 [PD-1] expressing the cell line MIT9 and human T cells) and in T cells from patients treated with nivolumab. Our method has potential for use as a simple and feasible monitoring system in the clinical setting.

Enzyme linked immunosorbent assay for the quantification of nivolumab and pembrolizumab in human serum and cerebrospinal fluid.

Immunotherapy with monoclonal antibodies targeting the programmed-death-1 (PD-1) receptor has become standard of care for an increasing number of tumor types. Pharmacokinetic studies may help to optimize anti-PD-1 therapy. Therefore, accurate and sensitive determination of antibody concentrations is essential. Here we report an enzyme linked immunosorbent assay (ELISA) capable of measuring nivolumab and pembrolizumab concentrations in serum and cerebrospinal fluid (CSF) with high sensitivity and specificity. The assay was developed and validated based on the specific capture of nivolumab and pembrolizumab by immobilized PD-1, with subsequent enzymatic chemiluminescent detection by anti-IgG4 coupled with horse radish peroxidase (HRP). The lower limit of quantification for serum and CSF was 2 ng/mL for both anti-PD-1 agents. The ELISA method was validated and showed long term sample stability of >1 year. This method is reliable, relatively inexpensive and can be used in serum and CSF from pembrolizumab and nivolumab treated patients.

High-resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method.

The determination of mAb critical quality attributes (CQA) is crucial for their successful application in health diseases. A generic CZE method was developed for the high-resolution separation of various mAb charge variants, which are often recognized as important CQA. A dynamic coating of the capillary was obtained with polyethylene oxide (PEO), whereas Bis-Tris allowed the analysis of mAbs under native conditions at pH 7.0. The effect of PEO and Bis-Tris concentrations, as well as the nature of the acidic counter ion on the method performance was systematically studied. The %RSD on migration times was below 5% on three different CE instruments using the optimized method. Additional charge variants (in particular acidic variants) were resolved for 10 out of 17 mAbs compared to a reference CZE approach involving the use of ε-amino-caproic acid (EACA), triethylenetetramine (TETA), and hydroxypropylmethyl cellulose (HPMC). The amount of basic and acidic charge variants of 17 Food and Drug Administration (FDA) approved mAbs covering a wide range of physico-chemical properties, e.g., pI between 8.0 and 9.4 and different hydrophobicity, were mainly comprised between 5-15% and 15-30%, respectively. It is noteworthy that applications for the quality control in hospitals as well as for the combination of the immune checkpoint inhibitors nivolumab and ipilimumab were presented.

Development of an Enzyme-Linked Immune Sorbent Assay to Measure Nivolumab and Pembrolizumab Serum Concentrations.

BACKGROUND: Treatment with monoclonal antibodies (mAbs) against programmed cell death protein 1 receptor is subject to high variation in treatment outcome among cancer patients. For these agents, no exposure-response (ER) relationships have been investigated in routine health care settings. However, ER relationships have been identified for several other mAbs used in oncology. Methods to conveniently measure serum concentrations of anti-programmed cell death protein 1 mAbs in routine health care may clarify possible ER relationships. Therefore, the authors aimed to develop an enzyme-linked immune sorbent assay (ELISA) for the measurement of both nivolumab and pembrolizumab serum concentrations of treated cancer patients. METHODS: Optimal capture antigen and detection antibody concentrations were selected based on titrations. Nivolumab calibration standards ranging from 0.2 to 300 ng/mL were tested in duplicate. Accuracy was assessed in 2 recovery experiments. Intra- and interassay variations were assessed on 3 different days by 2 independent technicians. The developed ELISA was also set up for pembrolizumab calibration curves. Cross-reactivity of nivolumab measurements with ipilimumab was assessed. Of one nivolumab treated patient, serum concentrations in follow up samples were measured and presented. RESULTS: Nivolumab calibration standards of 0.20-25 ng/mL were used. Nivolumab trough concentrations after 1 cycle in 8 patients ranged from 17.3 to 31.1 mcg/mL. The range of accuracy was 84%-105%, whereas intra- and interassay variations showed a coefficient of variation of 5.5% and 10.1%, respectively. No cross-reactivity with ipilimumab was detected. Pembrolizumab trough concentrations (n = 8) ranged from 9.1 to 19.7 mcg/mL after 1 infusion. CONCLUSIONS: The in-house-developed ELISA provides the opportunity to measure both nivolumab and pembrolizumab serum concentrations. This may help identify possible ER relationships in treated cancer patients and may potentially lead to dose adjustments in the future.