Comprehensive Analysis of Nivolumab, A Therapeutic Anti-Pd-1 Monoclonal Antibody: Impact of Handling and Stress

This study was partially funded by Project P20-01029 (I+D+i-Junta de Andalucia, Spain) and by Project B-FQM-308-UGR20 (Universidad de Granada, Proyectos I+D+i del Programa Operativo FEDER Andalucia 2020) which means that it was also partially supported by European Regional Development Funds.Nivolumab, formulated in the medicine Opdivo (R) (10 mg/mL), is a therapeutic monoclonal antibody (mAb) used in the treatment of different types of cancer. Currently, there is insufficient knowledge about the behaviour of this protein with regards to the risk associated with its routine handling or unintentional mishandling, or when subjected to stress conditions in hospitals. These conditions can be simulated in forced degradation studies, which provide an in-depth understanding of the biophysical and biochemical properties of mAbs. In this study, we carried out a physicochemical and functional characterisation of nivolumab, which was subjected to various stress conditions: heat, freeze/thaw cycles, agitation, light exposure and high hypertonic solution. We used a wide range of analytical techniques: Far-UV CD, IT-FS, DLS, SE/UHPLC(UV)-[Native]MS, and ELISA. The results show that exposure to light was the stress test with the greatest impact on the samples, revelling the formation of non-natural dimers and a different isoform profile. In addition, nivolumab (Opdivo (R)) demonstrated stability up to 60 degrees C (1 h). As regards functionality all the nivolumab (Opdivo (R)) stressed samples were found to be stable except for those subjected to light and agitation, and to a lesser extent, those subjected to FTC 5 and NaCl stresses.I+D+i-Junta de Andalucia, Spain P20-01029European Commission B-FQM-308-UGR2

The Relevance of Monoclonal Antibodies in the Treatment of COVID-19

This study was partially funded by Project FIS: PI-17/00547 (Institute of Health 'Carlos III', Ministry of Economy and Competitiveness, Spain), which means that it was also partially supported by European Regional Development Funds (ERDF).Major efforts have been made in the search for effective treatments since the outbreak of the COVID-19 infection in December 2019. Extensive research has been conducted on drugs that are already available and new treatments are also under development. Within this context, therapeutic monoclonal antibodies (mAbs) have been the subject of widespread investigation focusing on two target-based groups, i.e., non-SARS-CoV-2 specific mAbs, that target immune system responses, and SARS-CoV-2 specific mAbs, designed to neutralize the virus protein structure. Here we review the latest literature about the use of mAbs in order to describe the state of the art of the clinical trials and the benefits of using these biotherapeutics in the treatment of COVID-19. The clinical trials considered in the present review include both observational and randomized studies. We begin by presenting the studies conducted using non-SARS-CoV-2 specific mAbs for treating different immune disorders that were already on the market. Within this group of mAbs, we focus particularly on anti-IL-6/IL-6R. This is followed by a discussion of the studies on SARS-CoV-2 specific mAbs. Our findings indicate that SARS-CoV-2 specific mAbs are significantly more effective than non-specific ones.Institute of Health 'Carlos III', Ministry of Economy and Competitiveness, Spain - European Regional Development Funds (ERDF) FIS: PI-17/0054

Combined use of UV and MS data for ICH Stability-Indication Method: Quantification and isoforms identification of intact nivolumab

Nivolumab (Opdivo®) is a fully human immunoglobulin G4 isotype approved for the treatment of many cancers. It acts as an immune checkpoint inhibitor by blocking the interaction between PD-1 (Programmed Cell Death Protein 1) – an inhibitory receptor expressed on activated T cells- and its ligands, PD-L1 and PD-L2. The quantification of therapeutic proteins in their medicines and pharmaceutical preparations remains challenging because the protein content, a critical quality attribute, must be rigorously calculated using a validated stabilityindicating method, such as that indicated by the International Conference on Harmonization (ICH) quality guidelines, and this requires the analysis of the drug in the presence of its degraded products. In this work, we present an strategy based on the combined use of the UV and MS data to full file the requirement of the ICH-Q2 (R1) to develop and validated as stability indicated a (RP)UHPLC/UV-(HESI/Orbitrap™)MS method for the quantification of nivolumab in medicinal products. A comparative study of all figures of merit of the method using UV or MS data are shown and discussed. The results show that linearity was similar for the two detectors and was established over a range of 4–45 μg/mL and 1–45 μg/mL for the UV and (HESI/Orbitrap™)MS signals, respectively. The sensitivity of the method was higher when using the (HESI/Orbitrap™)MS signal (0.2 μg/mL) than with the UV(2.0 μg/mL). However, the UV signal provided better accuracy and precision than the (HESI/ Orbitrap™)MS signal, which did not meet the criteria for method robustness and system suitability. In spite of this, the MS signal plays a crucial role in this methodology by obtaining the molecular weight profile of the nivolumab isoforms, so enabling us to propose the glycans profile and detect structural modification due to degradation. The specificity of the method was evaluated by conducting forced degradation tests on samples of nivolumab in medicine form. The aim was to find out whether nivolumab suffers structural modifications when subject to stress. Structural modifications were detected by analysing the MS isoform profile, as changes of this kind promote new isoforms that are not chromatographically separated or detected by the UV signal. In this way, we demonstrated that the (RP)UHPLC/UV-(HESI/Orbitrap™)MS method was capable of detecting nivolumab degradation, and was suitable for use in nivolumab stability studies. Thus, the protein content in the daily surplus of the Opdivo® medicine, stored either at room temperature (20 ◦C) or refrigerated at 4 ◦C, could be tracked for 15 days.FPU18/03131 Ministry of Universities, Spain(P20_01029) Junta de Andalucía (Spain) and European Regional Development Fundspostdoctoral position from the Junta de Andalucía, SpainProject P20-01029 (I + D + i - Junta de Andalucía, Spain) Project B-FQM-308-UGR20 (Universidad de Granada, Proyectos I + D + i del Programa Operativo FEDER Andalucía 2020)CBUA/ Universidad de Granad

Comprehensive physicochemical characterization of a peptide-based medicine: Teduglutide (Revestive®) structural description and stress testing

Physico-chemical characterization; Stress testing; TeduglutideCaracterització fisicoquímica; Proves d'estrès; TeduglutidaCaracterización físicoquímica; Pruebas de estrés; TeduglutidaTeduglutide (Revestive®) is a glucagon-like peptide-2 analogue used for the treatment of short bowel syndrome, a rare life-threatening condition in which the amount of functional gut is too short to enable proper absorption of nutrients and fluids. During handling prior to administration to the patient in hospital, it is possible that peptide-based medicines may be exposed to environmental stress conditions that could affect their quality. It is therefore essential to carry out stress testing studies to evaluate how such medicines respond to these stresses. For this reason, in this paper we present a strategy for a comprehensive analytical characterization of a peptide and a stress testing study in which it was subjected to various stress conditions: heating at 40 °C and 60 °C, light exposure and shaking. Several complementary analytical techniques were used throughout this study: Far UV circular dichroism, intrinsic protein fluorescence spectroscopy, dynamic light scattering, size-exclusion chromatography and intact and peptide mapping reverse-phase chromatography coupled to mass spectrometry. To the best of our knowledge, this is the first study to offer an in-depth description of the chemical structure of teduglutide peptide and its physicochemical characteristics after stress stimuli were applied to the reconstituted medicine Revestive®.This study was funded by own funds from TEC01 (Ibs. Granada) and FQM-118 (University of Granada) research groups from University of Granada, and by the Hospital Paediatrics Pharmacy Unit of the Hospital Vall d’ Hebron (Barcelona, Spain), which supplied all the medicine samples. Raquel Pérez-Robles currently holds a postdoctoral position granted by the Junta de Andalucía, Spain (ref: DOC_01694). Jesús Hermosilla is currently benefiting from a research contract (Project ref: P20_01029) from the Junta de Andalucía (Spain) and European Regional Development Funds. Anabel Torrente-López is currently receiving an FPU predoctoral grant (ref.: FPU18/03131) from the Ministry of Universities, Spain. The authors would like to thank the two reviewers of this work for their feedback in revising this paper, which has resulted in a significant improvement of it. Funding for open access charge: Universidad de Granada / CBUA

Estabilidad en uso de los anticuerpos monoclonales terapéuticos tocilizumab, nivolumab y pembrolizumab: análisis comprensivo de parámetros fisicoquímicos y funcionalidad

En la presente Tesis Doctoral se han desarrollado, puesto a punto y validado estrategias analíticas comprensivas para evaluar distintos atributos críticos de la calidad de anticuerpos monoclonales terapéuticos (mAbs) comerciales. Con la finalidad de alcanzar los objetivos propuestos, estas estrategias se han empleado posteriormente en estudios de caracterización (fisicoquímica y funcional) y estabilidad de los mismas en condiciones de uso hospitalario, los cuales se recogen en los distintos capítulos que conforman esta Tesis Doctoral. Los principales mAbs estudiados han sido tocilizumab, nivolumab y pembrolizumab, aunque también se ha realizado un estudio que recoge una amplia selección de mAbs terapéuticos. Los trabajos recogidos en esta Tesis Doctoral se pueden dividir en tres grandes secciones. La primera sección recoge dos trabajos relacionados con mAbs empleados en el tratamiento de la COVID-19. Esto se debe a que el inicio de esta Tesis coincidió con el inicio de la pandemia causada por el coronavirus SARS-CoV-2, en octubre de 2019. En el primer capítulo se presenta un trabajo de revisión bibliográfica de los mAbs empleados en el tratamiento de la COVID- 19, en el que se ha hecho una diferenciación entre los mAbs no específicos y específicos del SARS-CoV-2. El primer grupo recoge aquellos mAbs que bloquean la acción de la interleucina-6 (IL-6), la cual provoca una respuesta inmune exagerada en los pacientes con dicha enfermedad, por lo que estos mAbs van dirigidos a bloquear un punto clave de la respuesta del sistema inmune de los pacientes infectados. En cambio, el segundo grupo contempla aquellos mAbs que son capaces de neutralizar directamente la estructura proteica del virus, donde se recogen, además, los mAbs que recibieron una autorización de uso de emergencia. El segundo capítulo recoge un trabajo en el que se han estudiado los atributos fisicoquímicos de la calidad de un mAb empleado en el tratamiento de la COVID-19, tocilizumab (RoActemra®, Roche Registration GmbH, Alemania), el cual se trata de un inhibidor de la IL-6. En este estudio se comparan los principales atributos fisicoquímicos de las formas farmacéuticas intravenosa (IV) y subcutánea (SC) de dicho medicamento, con el objetivo de averiguar si la forma farmacéutica SC podía ser administrada de manera IV como consecuencia del desabastecimiento hospitalario de la forma IV. Para ello se utilizaron una amplia variedad de técnicas analíticas: dispersión de luz dinámica (DLS), fluorescencia intrínseca de triptófanos (ITFS), cromatografía de exclusión molecular (SEC) y cromatografía de intercambio catiónico (CEX). Estas técnicas permitieron comparar ambas formas medicamentosas a las concentraciones de disolución clínica de 6 y 4 mg/mL de tocilizumab. Además, también se llevaron a cabo estudios de degradación controlada con el objetivo de corroborar la estabilidad de todos los métodos utilizados y para evaluar la comparabilidad de las disoluciones clínicas IV y SC. La segunda sección de esta Tesis está compuesta por tres trabajos llevados a cabo con mAbs inhibidores de puntos de control inmunitario, concretamente estos mAbs tienen la capacidad de bloquear la acción del receptor PD-1. En primer lugar, el tercer capítulo recoge un trabajo realizado con nivolumab (Opdivo®, Bristol-Myers Squibb Pharma EEIG, Irlanda) en el que se ha desarrollado y validado un método cromatográfico de fase inversa con detección ultravioleta y por espectrometría de masas ((RP)UHPLC/UV- (HESI/Orbitrap™)MS) para la cuantificación del contenido proteico de dicho medicamento. Este se trata de un método indicador de la estabilidad capaz de analizar el fármaco en presencia de sus productos degradados. Tanto el método de detección por absorción en el ultravioleta como el método utilizando espectrometría de masas han sido validados mediante el estudio de determinados parámetros de calidad del método, tales como linealidad, límites de detección y cuantificación, precisión, exactitud, especificidad, robustez y test de la adecuación al sistema (system suitability test). La especificidad del método ha sido evaluada mediante ensayos de degradación forzada en muestas de Opdivo®. Este trabajo también recoge la identificación del perfil de isoformas de nivolumab gracias a la señal obtenida por espectrometría de masas de alta resolución y masa exacta. Además, se realizó un estudio de estabilidad del medicamento durante 15 días para evaluar la aplicabilidad del método. En cuanto al cuarto capítulo, este recoge un análisis comprensivo de las características fisicoquímicas y funcionales de nivolumab (Opdivo®). Para ello, se utilizaron diversas y complementarias técnicas analíticas que aportan información sobre distintos aspectos fisicoquímicos de la proteína, tales como su estructura secundaria y terciaria, el perfil de agregación o el estudio de sus isoformas. Las técnicas utilizadas se pueden agrupar en técnicas espectrofotométricas (dicroísmo circular (CD), IT-FS y DLS), cromatográficas (SEC) y espectrometría de masas. La evaluación de la funcionalidad se llevó a cabo utilizando la técnica ELISA. También se llevaron a cabo estudios de degradación forzada para estudiar en profundidad las propiedades del medicamento, para lo que se sometieron muestras de Opdivo® a distintas condiciones estresantes, tales como elevada temperatura, ciclos de congelación/descongelación, agitación, exposición a la luz y exposición a una alta concentración salina. Los métodos fueron desarrollados ad hoc y validados. El último capítulo de esta sección, el capítulo 5, recoge un trabajo de estructura similar al del capítulo 4, en este caso llevado a cabo con pembrolizumab (Keytruda®, Merck Sharp & Dohme B.V., Países Bajos). En él se ha realizado un análisis comprensivo del medicamento mencionado mediante su caracterización fisicoquímica y funcional a través del empleo de distintas técnicas analíticas complementarias. Entre las técnicas utilizadas encontramos CD, IT-FS, DLS, SEC, RP/UHPLC(UV)-MS/MS y ELISA. Estas han permitido el estudio de las estructuras de orden superior de la proteína, la formación de particulado y agregación, el estudio de las isoformas y la determinación de las modificaciones postraduccionales. En este trabajo también se llevaron a cabo estudios de degradación forzada de muestras del medicamento, el cual fue sometido a diversas condiciones estresantes, tales como altas temperaturas, ciclos de congelación/descongelación, exposición a la luz, exposición a agitación fuerte y suave y exposición a una alta concentración salina. Todas las muestras sometidas a estos estreses fueron analizadas mediante la metodología desarrollada ad hoc y validada empleando las técnicas analíticas mencionadas; los resultados fueron siempre comparados con los resultados de la muestra control del medicamento, es decir, una muestra no sometida a ningún tipo de estrés. De esta forma, los trabajos recogidos en los capítulos 4 y 5 evalúan el impacto de la manipulación hospitalaria sobre la estabilidad de los medicamentos Opdivo® y Keytruda®, y ofrecen recomendaciones sobre el correcto manejo de los mismos durante su manipulación. Por último, la tercera sección está formada por el último capítulo de esta Tesis, capítulo 6, el cual recoge un trabajo realizado sobre un total de diez mAbs terapéuticos comerciales diferentes. Este trabajo consistió en la optimización del ensayo ReFOLD, un método isotérmico que permite predecir la estabilidad a largo plazo de medicamentos basados en mAbs mediante un proceso de desnaturalización química con urea. Para ello, se sometieron los distintos mAbs seleccionados (a tres concentraciones diferentes cada uno) a dos concentraciones altas y distintas de urea (6 M y 6.5 M). Los resultados obtenidos fueron comparados con los resultados de ensayos previos realizados con una concentración de urea de 10 M, con el objetivo de mejorar las dificultades encontradas con tan alta concentración de urea, ya que la concentración de 10 M mostró efectos indeseados en varios de los productos farmacéuticos ensayados, tales como la formación de precipitados. Además, también se estudió la capacidad de mantener una clasificación de la estabilidad de los productos farmacéuticos ensayados con las nuevas concentraciones de urea. Como técnica analítica, en este caso, se empleó SEC para estudiar la capacidad de recuperación del estado nativo de la proteína tras ser sometida a las altas concentraciones de urea.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.Tesis Univ. Granada.FPU predoctoral grant (ref.: FPU18/03131

Use of subcutaneous tocilizumab to prepare intravenous solutions for COVID-19 emergency shortage: Comparative analytical study of physicochemical quality attributes

COVID-19, a disease caused by the novel coronavirus SARS-CoV-2, has produced a serious emergency for global public health, placing enormous stress on national health systems in many countries. Several studies suggest that cytokine storms (interleukins) may play an important role in severe cases of COVID19. Neutralizing key inflammatory factors in cytokine release syndrome (CRS) could therefore be of great value in reducing the mortality rate. Tocilizumab (TCZ) in its intravenous (IV) form of administration -RoActemra® 20 mg/mL (Roche)-is indicated for treatment of severe CRS patients. Preliminary investigations have concluded that inhibition of IL-6 with TCZ appears to be efficacious and safe, with several ongoing clinical trials. This has led to a huge increase in demand for IV TCZ for treating severe COVID-19 patients in hospitals, which has resulted in drug shortages. Here, we present a comparability study assessing the main critical physicochemical attributes of TCZ solutions used for infusion, at 6 mg/ mL and 4 mg/mL, prepared from RoActemra® 20 mg/mL (IV form) and from RoActemra® 162 mg (0.9 mL solution pre-filled syringe, subcutaneous(SC) form), to evaluate the use of the latter for preparing clinical solutions required for IV administration, so that in a situation of shortage of the IV medicine, the SC form could be used to prepare the solutions for IV delivery of TCZ. It is important to remember that during the current pandemic all the medicines are used off-label, since none of them has yet been approved for the treatment of COVID-19.Instituto Carlos III, Ministerio de Economia y Competitividad, Spain FIS: PI-17/00547European Union (EU)Ministry of Universities, Spain FPU18/03131University of Granada (Spain