In-use stability of the therapeutic monoclonal antibodies Tocilizumab, Nivolumab and PembrolizumabComprehensive analysis of physicochemical parameters and functionality

Abstract

In this Doctoral Thesis, comprehensive analytical strategies have been developed and validated to evaluate different critical quality attributes of commercial therapeutic monoclonal antibodies (mAbs). In order to achieve the proposed objectives, these strategies have subsequently been used in characterisation studies (physicochemical and functional) and stability studies under hospital use conditions, which are included in the different chapters of this Doctoral Thesis. The main mAbs studied were tocilizumab, nivolumab and pembrolizumab, although a study was also carried out on a wide selection of therapeutic mAbs. The works included in this doctoral thesis can be divided into three main sections. The first section includes two scientific contributions related to mAbs used in the treatment of COVID-19. This is because the start of this Thesis coincided with the beginning of the pandemic caused by the SARS-CoV-2 coronavirus, in October 2019. The first chapter presents a review of the mAbs used in the treatment of COVID-19, in which a differentiation has been made between non-specific and specific mAbs for SARS-CoV-2. The first group includes mAbs that block the action of interleukin-6 (IL-6), which causes an exaggerated immune response in patients with the disease, so these mAbs are aimed at blocking a key point in the immune system response of infected patients. In contrast, the second group includes mAbs that are capable of directly neutralising the protein structure of the virus, including mAbs that have received emergency use authorisation. The second chapter is a study of the physicochemical quality attributes of a mAb used in the treatment of COVID-19, tocilizumab (RoActemra®, Roche Registration GmbH, Germany), which is an IL-6 inhibitor. This study compares the main physicochemical attributes of the intravenous (IV) and subcutaneous (SC) dosage forms of the medicine, with the aim of finding out whether the SC dosage form could be administered as IV because of hospital shortage of the IV form. A wide variety of analytical techniques were used: dynamic light scattering (DLS), intrinsic tryptophan fluorescence (IT-FS), molecular exclusion chromatography (SEC) and cation exchange chromatography (CEX). These techniques allowed comparison of both drug forms at clinical dilution concentrations of 6 and 4 mg/mL of tocilizumab. In addition, controlled degradation studies were also performed to corroborate the stability of all methods used and to assess the comparability of the IV and SC clinical dilutions. The second section of this Thesis is composed of three studies carried out with immune checkpoint inhibitor mAbs, specifically these mAbs have the capacity to block the action of the PD-1 receptor. Firstly, the third chapter includes a study carried out with nivolumab (Opdivo®, Bristol-Myers Squibb Pharma EEIG, Ireland) in which a reverse-phase chromatographic method with ultraviolet detection and mass spectrometry ((RP)UHPLC/UV-(HESI/Orbitrap™)MS) has been developed and validated for the quantification of the protein content of this drug. This is a stability indicator method capable of analysing the drug in the presence of its degraded products. Both the ultraviolet absorption detection method and the method using mass spectrometry have been validated by studying certain method quality parameters, such as linearity, detection and quantification limits, precision, accuracy, specificity, robustness and system suitability test. The specificity of the method has been evaluated by forced degradation tests on Opdivo® samples. This work also includes the identification of the nivolumab isoform profile thanks to the signal obtained by high-resolution and accurate mass spectrometry. In addition, a 15-day medicine stability study was performed to assess the applicability of the method. The fourth chapter contains a comprehensive analysis of the physicochemical and functional characteristics of nivolumab (Opdivo®). To this end, various complementary analytical techniques were used to provide information on different physicochemical aspects of the protein, such as its secondary and tertiary structure, aggregation profile or the study of isoforms. The techniques used can be grouped into spectrophotometric (circular dichroism (CD), IT-FS and DLS), chromatographic (SEC) and mass spectrometric techniques. Functionality assessment was carried out using the ELISA technique. Forced degradation studies were also carried out to study in depth the properties of the medicine by subjecting Opdivo® samples to different stressful conditions such as high temperature, freeze/thaw cycles, agitation, exposure to light and exposure to high salt concentration. The methods were developed ad hoc and validated. The last chapter of this section, chapter 5, contains a study with a similar structure to chapter 4, in this case carried out with pembrolizumab (Keytruda®, Merck Sharp & Dohme B.V., The Netherlands). A comprehensive analysis of pembrolizumab has been carried out by physicochemical and functional characterisation using different complementary analytical techniques. Techniques used include CD, IT-FS, DLS, SEC, RP/UHPLC(UV)-MS/MS and ELISA. These have allowed the study of the higher order structures of the protein, particle formation and aggregation, the study of isoforms and the determination of post-translational modifications. In this work, forced degradation studies were also carried out on samples of the medicine, which was subjected to various stressful conditions, such as high temperatures, freeze/thaw cycles, exposure to light, exposure to strong and gentle agitation, and exposure to high salt concentration. All samples subjected to these stresses were analysed using the methodology developed ad hoc and validated using the abovementioned analytical techniques; the results were always compared with the results of the control sample of the medicine, i.e., a sample not subjected to any stress. Thus, the works in chapters 4 and 5 assess the impact of hospital handling on the stability of Opdivo® and Keytruda® medicines, and provide recommendations on the correct handling of these medicines during their manipulation. Finally, the third section presents the last chapter of this Thesis, chapter 6, which includes a work carried out on a total of ten different commercial therapeutic mAbs. This work consisted of the optimisation of the ReFOLD assay, an isothermal method that allows predicting the long-term stability of mAb-based drugs by means of a chemical denaturation process with urea. For this purpose, selected mAbs (at three different concentrations each) were subjected to two different high concentrations of urea (6 M and 6.5 M). The results obtained were compared with the results of previous tests carried out with a urea concentration of 10 M, with the aim of improving the difficulties encountered with such a high urea concentration, since the 10 M concentration showed undesired effects on several of the tested pharmaceuticals, such as the formation of precipitates. In addition, the ability to maintain a stability ranking of the tested pharmaceuticals with the new urea concentrations was also studied. As an analytical technique, in this case, SEC was used to study the recovery capacity of the native state of the protein after being subjected to the high urea concentrations.