Extravasation accidents with liposomal/liposomal pegylated anthracyclines treated with dexrazoxane: an overview and outcomes.

The extravasation of chemotherapeutic agents is a challenge for oncologic care teams. The management of nonliposomal (conventional) anthracyclines is well established in clinical practice guidelines, including general measures and specific antidotes, such as dexrazoxane. However, there is little scientific evidence on the management of liposomal and pegylated liposomal anthracyclines. The aim of this paper was to review the scientific literature on the extravasation of liposomal and pegylated liposomal anthracyclines and determine the clinical impact of this type of extravasation, focusing on dexrazoxane. The literature was searched using two databases: PubMed and Embase. Three searches were conducted, using liposomal anthracycline extravasation, pegylated liposomal anthracycline extravasation, and liposomal doxorubicin extravasation as keywords, respectively. Seven articles fulfilled the study eligibility criteria and included seventeen cases in humans. Extravasation occurred with three drugs: liposomal doxorubicin in nine (53%) patients, liposomal daunorubicin in four (23.5%) patients, and pegylated liposomal doxorubicin in four (23.5%) patients. General measures for extravasations were applied in all patients, but only three patients received dexrazoxane. All cases were completely resolved at 2-3 months, except for one patient, in whom dexrazoxane was not used. In animals, dexrazoxane decreased both the frequency of wounds produced by pegylated liposomal doxorubicin and their extent. The pharmacokinetic profiles of liposomal and pegylated liposomal anthracyclines differ from those of conventional anthracyclines, modifying their effectiveness and safety. General measures may be inadequate to heal areas affected by extravasation, which may require the administration of dexrazoxane. However, each case should be evaluated individually for the administration of dexrazoxane in off-label use until scientific evidence is available on its effectiveness and safety as an antidote for these formulations of anthracyclines.

Cluster-based comparison of the peptide mass fingerprint obtained by MALDI-TOF mass spectrometry. A case study: long-term stability of rituximab.

We evaluated the use of the peptide mass fingerprint (PMF) obtained by matrix assisted laser desorption and ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) to track changes in the structure of a protein. The first problem we had to overcome was the inherent complexity of the PMF, which makes it difficult to compare. We dealt with this problem by developing a cluster-based comparison algorithm which takes into account the proportional error made by the mass spectrometer. This procedure involves grouping together similar masses in an intelligent manner, so that we can determine which data correspond to the same peptide (any slight differences can be explained as experimental errors), and which of them are too different and thus more likely to represent different peptides. The proposed algorithm was applied to track changes in a commercially available monoclonal antibody (mAb), namely rituximab (RTX), prepared under the usual hospital conditions and stored refrigerated (4 °C) and frozen (-20 °C) for a long term study. PMFs were obtained periodically over three months. For each checked time, five replicates of the PMFs were obtained in order to evaluate the similarities between them by means of the occurrences of the particular peptides (m/z). After applying the algorithm to the PMF, different approaches were used to analyse the results. Surprisingly, all of them suggested that there were no differences between the two storage conditions tested, i.e. the RTX samples were almost equally well preserved when stored refrigerated at 4 °C or frozen at -20 °C. The cluster-based methodology is new in protein mass spectrometry and could be useful as an easy test for major changes in proteins and biopharmaceutics for diverse applications in industry and other fields, and could provide additional stability data in relation to the practical use of anticancer drugs.

Comprehensive physicochemical and functional analysis of pembrolizumab based on controlled degradation studies: Impact on antigen-antibody binding.

Monoclonal antibodies-based medicines are widely used in the treatment of different diseases. These medicines are very sensitive to exposure to different environmental conditions and their handling in hospitals may affect their safety and efficacy. This is the case for pembrolizumab (Keytruda®, 25 mg/mL), for which there is not yet much information on its risk behaviour associated with routine handling or unintentional mishandling. Here we performed a wider physicochemical and functional analysis of pembrolizumab medicine including controlled degradation studies: heat, freeze/thaw, agitation, accelerated light exposure and high hypertonic solution. After that, the samples were analysed by a set of analytical techniques to evaluated critical quality attributes: Far-UV CD, IT-FS, DLS, RP/UHPLC(UV)-MS, SE/UHPLC(UV), RP/UHPLC(UV)-MS/MS and ELISA. The results provide an in-depth understanding of the biochemical and biophysical properties of pembrolizumab, showing that the medicine is affected by accelerated light exposure and temperature of 60 °C, demonstrated by the detection of non-natural dimers and HMWS. Light exposure also revealed different isoform profile and increase in oxidations. Regarding functionality by means of the interaction antigen-antibody binding, all the stressors promoted a decrease in pembrolizumab capacity to bind to PD-1 receptor, although the biological activity remained still high for all of them, being 60 °C and accelerated light exposure the most affected.

Quantification of an intact monoclonal antibody, rituximab, by (RP)HPLC/DAD in compliance with ICH guidelines.

We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reversephase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stabilityindicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm×4.6 mm, 5 µm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ =214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines.We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.

Analysing the In-Use Stability of mRNA-LNP COVID-19 Vaccines Comirnaty™ (Pfizer) and Spikevax™ (Moderna): A Comparative Study of the Particulate.

Comirnaty™ and Spikevax™ were the first vaccines approved for human use based on modified non-replicating mRNA lipophilic nanoparticle (mRNA-LNP) technology, with great success in the treatment of COVID-19. They have been used massively worldwide. One of the major inconveniences of these vaccines is related to pharmaceutical stability issues. Proper transportation, storage, and in-use handling before administration to patients are critical steps since failures can potentially reduce potency. In this research, the in-use stability of Comirnaty™ and Spikevax™ clinical samples was analysed and the results were compared. As changes in the size of the mRNA-LNPs are related to potency, these modifications were analysed by qualitative Dynamic Light Scattering (DLS) as a stability-indicating method for control and stressed vaccine samples. Strong stress factors (accelerated light irradiation, manual shaking, and vortex vibration) and conditions that mimic in-use handling (exposure to natural light and room temperature, repeated cycles of injections, and 24 h storage in syringes) were checked. The morphology of the mRNA-LNPs was analysed by Transmission Electron Microscopy (TEM) to better interpret and support the DLS results. Although the two vaccines are based on the same mRNA-LNP technology, the results demonstrate that they are characterised by very different particle size profiles and behaviours against different handling/stress conditions.

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

Teduglutide (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®.

Degradation and in-use stability study of five marketed therapeutic monoclonal antibodies by generic weak cation exchange liquid chromatographic method ((WCX)HPLC/DAD).

Therapeutic monoclonal antibodies (mAbs) represent a very important class of the current biopharmaceutics. The great complexity of their structure made necessary the use of different analytical approaches for assessing different physico-chemical properties. In this work, weak cation exchange (WCX) high performance liquid chromatography with diode array detection ((WCX)HPLC/DAD) is used to assess the charge variant profile. The method here developed combined the effect of ionic strength and controlled pH gradient and allows for the charge variants analysis of the five mAbs studied, namely bevacizumab (BVZ), cetuximab (CTX) infliximab (INF), rituximab (RTX) and trastuzumab (TTZ), which are among the most used mAbs worldwide. The differences in the charge variants in the natural isoforms of the mAbs promoted characteristic WCX chromatograms for each of mAbs that can be also useful for identification purposes. These chromatograms have provided to be suitable for tracking changes in the charge variants of each mAb analyzed both in controlled degraded and in stabilities study along time of in-use samples solutions at 2 mg/mL in 0.9% NaCl stored refrigerated (at 4 °C) and frozen (-20 °C) for two months. The results obtained indicated different stabilities of these mAbs, all IgG1, against degradation by different stressed environmental conditions and in-use stability along two months.

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

Nivolumab, formulated in the medicine Opdivo® (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®) demonstrated stability up to 60 °C (1 h). As regards functionality all the nivolumab (Opdivo®) 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.

Method for identification and quantification of intact teduglutide peptide using (RP)UHPLC-UV-(HESI/ORBITRAP)MS.

Teduglutide (Revestive®, 10 mg mL-1) is a recombinant human glucagon-like peptide 2 analogue, used in the treatment of short bowel syndrome, a serious and highly disabling condition which results from either too small a length of intestine or loss of critical intestinal function. The determination of therapeutic compounds of protein-nature is always challenging due to their complex structure. In this work, we present a fast, straightforward reversed phase (RP)UHPLC-UV-(HESI/ORBITRAP)MS method for the identification and quantification of the intact teduglutide peptide. The method has been developed and validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines; therefore, linearity, limits of detection and quantification, accuracy (precision and trueness), robustness, system suitability and specificity using the signal from the UV and MS, have been evaluated. The validation performance parameters obtained from the UV and MS signals were compared throughout the work, to select the most suitable. To study the specificity of the method and the impact of medicine mishandling under hospital conditions, force degradation studies were performed, i.e. thermal (40 °C and 60 °C), shaking (mechanical) and light (accelerated exposition) effects. Identification by the exact mass of teduglutide was achieved and it was confirmed that the peptide does not undergo any post-translational modifications (PTMs). To the best of our knowledge, the present work reports the first method developed for the simultaneous identification, structural characterization, and quantification of the therapeutic teduglutide peptide. Finally, the proposed method is able to indicate stability when quantifying the intact teduglutide since detects and characterises the exact mass of the degradation/modification products.

Tracking the physicochemical stability of teduglutide (Revestive®) clinical solutions over time in different storage containers.

Teduglutide, the active ingredient of the medicine Revestive® (5 mg), is a recombinant therapeutic peptide that mimics the effects of the endogenous glucagon-like peptide 2 (GLP-2). It stimulates intestinal growth, adaptation and function in patients with Short Bowel Syndrome who are dependent on parenteral nutrition. The Summary of Product Characteristics recommends immediate use of the reconstituted solutions and the discarding of any subsequent surplus. This study aims to carry out a long-term stability study that reproduces hospital conditions of use which provide sound evidence regarding the use of teduglutide surplus beyond the Summary Product Characteristics recommendations. We conducted a stability study of teduglutide solutions prepared from a 5 mg vial of Revestive®. Some of the solutions were stored in their original vial after reconstitution, while others were repackaged in plastic syringes to evaluate their physicochemical stability over time. For this purpose, we applied a set of previously validated analytical methodologies to evaluate the main critical quality attributes of teduglutide, i.e., primary (including post-tralational modifications), secondary and tertiary structures, aggregates, particulate, concentration and pH. The results indicate that the solutions maintain high physicochemical stability over time, regardless of the storage temperature (4ºC or -20ºC) or the storage container (vials or syringes). This research provides new data on the stability of Revestive® that will be of great value to hospital pharmacists. This comprehensive assessment of the physicochemical long-term stability of TGT has demonstrated that under the storage conditions and over the period studied here, the medicine maintains its quality, efficacy and safety profiles.

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

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.

Stability study over time of clinical solutions of ziv-aflibercept prepared in infusion bags using a proper combination of physicochemical and functional strategies.

A range of biopharmaceutical products are used to target Vascular Endothelial Growth Factor (VEGF), including Eylea® (aflibercept, AFL) and Zaltrap® (ziv-aflibercept, ziv-AFL). The first is indicated for ophthalmological diseases such as neovascular (wet) age-related macular degeneration, while the second is used in the treatment of metastatic colorectal cancer. The stability of AFL in prefilled syringes has been widely studied; however, no research has yet been done on the stability of ziv-AFL in polyolefin infusion bags. Therefore, the purpose of the present research is to evaluate the stability of ziv-AFL (Zaltrap®) clinical solutions prepared under aseptic conditions in polyolefin infusion bags at two different concentrations, i.e. 4.0 and 0.6 mg/mL, and stored refrigerated in darkness at 2-8 °C for 14 days. With that aim, the ziv-AFL clinical solutions were assessed by analysing changes in its physicochemical and functional properties. The distribution of the particulates was studied over a range of 0.001-10 µm by Dynamic Light Scattering (DLS); oligomers were analysed by Size-Exclusion High-Performance Chromatography with Diode Array Detection (SE/HLPC-DAD); the secondary structure of the protein was studied by far UV Circular Dichroism (CD) and the tertiary structure by Intrinsic Tryptophan Fluorescence (IT-F) and Intrinsic Protein Fluorescence (IP-F); charge variants were assessed by Strong Cation Exchange Ultra-High-Performance Chromatography with UV detection (SCX/UHPLC-UV); functionality was evaluated by ELISA by measuring the biological activity as manifested in the extension of the immunological reaction of the ziv-AFL with its antigen (VEGF). Neither aggregation nor oligomerization were detected by the techniques mentioned above. Secondary and tertiary structures remained unchanged over the 14-day period, as did charge variants. The functionality observed initially was maintained along time. Therefore, it could be proposed that the ziv-AFL clinical solutions studied showed great physicochemical and functional stability over a period of two weeks, regardless of the concentration, i.e. 4 or 0.6 mg/mL.

Detection of adverse drug reactions through the minimum basic data set.

OBJECTIVE: To analyze adverse drug reaction (ADR) detection using the Minimum Basic Data Set (MBDS) at hospital discharge and to compare the ADR reporting rate to the Pharmacovigilance Referral Centre with other similar hospitals that do not use this reporting system. Setting 650-bed University Hospital serving a population of 294,000 inhabitants in Spain. METHOD: A retrospective descriptive study was conducted between January 2006 and December 2007. All reports of ADRs gathered in MBDS (a tool that encodes all administrative and clinical information generated for each patient during a hospitalization episode) with International Classification Disease codes between E930 and E949.9 were analyzed to assess the appropriateness of their referral to the pharmacovigilance centre. Finally, we compared our reporting rate with other hospitals that do not use this system for ADR identification. MAIN OUTCOME MEASURE: The incidence of ADRs detected in hospitalized patients and the reporting rate (per thousand inhabitants) to the referral pharmacovigilance centre using the Yellow Card system. RESULTS: Out of 43,282 hospital discharges, 386 ADR were recorded (0.89% of hospitalized patients). The mean (+/-SD) age of patients with reported ADR was 61.9 years (+/-19.2), median age was 65 years, and 55.2% were female. The Department of Pharmacy reported 276 (71.5%) of ADR using the Yellow Card system. The most frequently reported drugs were anti-cancer agents (42.5%) and cardiovascular drugs (23.8%), with a high frequency of digitalis glycosides (18.4%). ADR were most frequently recorded by the Departments of Oncology (41.7%) and Internal Medicine (17.9%). CONCLUSION: The MBDS is a useful and accessible instrument to determine the incidence of ADR in a hospital, resulting in the notification of severe events that might otherwise not be reported. Its use also improves identification of the main drugs responsible for ADR and of the patient populations at greatest risk, facilitating the implementation of alert systems and the development of prevention and detection strategies.

Intact charge variant analysis of ziv-aflibercept by cationic exchange liquid chromatography as a proof of concept: Comparison between volatile and non-volatile salts in the mobile phase.

Ziv-aflibercept (ziv-AFL) is a complex fusion protein which is widely used in hospitals for the treatment of colorectal metastatic cancer. Charge variants are critical attributes for assessing post-transitional modifications (PMTs) that have to be controlled during the development and manufacture of these proteins and until their administration to patients. Cation exchange (CEX) chromatography is a charge-sensitive analytical method that is well suited for analysing charge variants in proteins. The aim of this paper is to analyse the charge variants of ziv-AFL in the medicine (Zaltrap®) when fresh and when degraded. Two CEX chromatographic methods were compared for this purpose. The former was an adaptation of the method used in the first published study in which charge variants were analysed via pH gradient elution using volatile, low ionic strength buffers with direct coupling to high-resolution Orbitrap mass spectrometry. The second method was developed and optimized during our research using the salt-mediated pH gradient mode and classical non-volatile, high ionic strength buffers which were incompatible with direct coupling with mass detection. Fresh and controlled degraded samples of ziv-AFL were used to evaluate the capacity of both CEX chromatographic strategies for detecting charge variants in ziv-AFL. In the controlled degradation study the samples of the medicine were subjected to three stress factors: temperature of 60 °C for three hours, freeze/thaw process -two cycles-, and exposure to light for twelve hours. The CEX chromatographic method with non-volatile salts in the mobile phase enabled better detection of charge variants degraded ziv-AFL samples than the method using volatile salts with lower ionic strength. In addition, the complexity of the mass spectra data generated made it impossible to identify the multicharge variant species of ziv-AFL. Although charge variants were not separated in ziv-AFL fresh sample, our results indicate that the method with non-volatile salts in the mobile phase could be used to characterize and track changes in the charge variant UV chromatographic profile of ziv-AFL in fresh and degraded samples, even though it cannot be coupled to a mass detector and there is therefore no information about mass. The increase of basic protein degraded compounds were the most important degradation pattern detected in ziv-AFL (Zaltrap®).

Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation.

Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases. There are two medicines in which AFL is the active substance: Zaltrap and Eylea, referred as ziv-AFL and AFL respectively. No proper accelerated degradation studies were published on either AFL or ziv-AFL. These studies are essential during research, development and manufacturing stages. Here, we characterized ziv-AFL and submitted it to different stress conditions: light, 60 °C, freeze-thaw cycles, changes in pH, high hypertonic solution and strong denaturing conditions. We used an array of techniques to detect aggregation (SE-HPLC/DAD and DLS), changes in secondary structure (Far-UV circular dichroism), changes in conformation or tertiary structure (Intrinsic tryptophan fluorescence) and alterations in functionality (ELISA). Results indicate that aggregation is common degradation pathway. Two different types of aggregates were detected: dimers and high molecular weight aggregates attributed to ß-amyloid-like structures. Secondary structure was maintained in most of the stress tests, while conformation was altered by almost all the tests except for the freeze-thaw cycles. Functionality, evaluated by its immunochemical reaction with VEGF, was found to be stable but with decrease when exposed to light and with likely partial inactivation of the drug when pH was altered.

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 COVID-19. 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.

Comparative Stability Studies of Different Infliximab and Biosimilar CT-P13 Clinical Solutions by Combined Use of Physicochemical Analytical Techniques and Enzyme-Linked Immunosorbent Assay (ELISA).

BACKGROUND: There are two products in which infliximab is the active pharmaceutical ingredient. These are Remicade® (INF; reference product) and Remsima™/Inflectra™ (CT-P13; infliximab biosimilar). Remsima™/Inflectra™ are bioidentical products. Different recommendations have been made for the clinical solutions of each brand (Remicade® or Remsima™/Inflectra™) despite the manufacturer of the biosimilar claiming high levels of similarity to the innovator. OBJECTIVE: The objective of this study was to assess and compare stability against degradation and over time of different clinical infliximab solutions prepared from Remicade® and from Remsima™/Inflectra™ using a suitable set of characterization methods in line with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) recommendations. METHODS: Reconstituted solutions of INF and CT-P13 and dilutions as used in hospital were stored in glass vials (10 and 2 mg/mL) or in polyolefin infusion bags (0.4 mg/mL) refrigerated between 2 and 8 °C for 2 weeks. Regarding the physicochemical properties, the distribution of the particulates were studied over a range of 0.001-1 µm by dynamic light scattering (DLS) and oligomers up to 8 monomer were analyzed by native size-exclusion ultra-high-performance liquid chromatography with ultraviolet (UV)-visible detection coupled to (native) mass spectrometry (SE/UHPLC-UV-(native) MS); mass spectrometry was also used to evaluate natural aggregates and isoform profile; DLS was also employed to detect gross conformational changes by tracking the hydrodynamic radius (HR). The secondary structure of the proteins was studied by far UV circular dichroism (CD). The tertiary structure was investigated by intrinsic tryptophan fluorescence (IT-F). Reverse-phase ultra-high-performance liquid chromatography with UV detection (RP/UHPLC-UV) was used to analyze intact INF and CT-P13 for quantification purposes. Functionality was evaluated via the biological activity measured by the extension of the immunological reaction of the INF and the CT-P13 with its antigen, i.e., the tumor necrosis factor-α by enzyme-linked immunosorbent assay (ELISA). RESULTS: The stress applied to INF and CT-P13 solutions showed similar levels of aggregate formation, structural variation, and chemical modifications. The only noteworthy difference between INF and CT-P13 was detected in their behavior to freeze-thaw cycles, in which CT-P13 showed slightly more robustness. INF and CT-P13 showed identical CD spectra, similar to those reported for IgG1 in which there is dominance in ß sheet secondary structures; this typical conformation remained unmodified over time in INF and CT-P13. No significant changes were detected in the tertiary structure and no aggregates process was noticed over the time studied. Polydispersity slightly increased for the most concentrated solutions, while there were no meaningful differences in the HR in the solutions over time. The concentration of INF and CT-P13 also remained constant. Differences in the native isoform MS profile were detected, as expected by the different glycosylation pattern, with no important modification over time. Functionality was maintained over the test period (60 days) and was similar in all the solutions tested, with no differences between INF and biosimilar solutions. CONCLUSIONS: High levels of similarity were noticed in the behavior of INF and CT-P13 when subjected to stress. When stored refrigerated at between 2 and 8 °C and prepared as normally used in the hospital pharmacy, all solutions showed physicochemical and functional stability for all the concentrations tested and all containers, at least for the 14-day test period.

Development and validation of a (RP)UHPLC-UV-(HESI/Orbitrap)MS method for the identification and quantification of mixtures of intact therapeutical monoclonal antibodies using a monolithic column.

Monoclonal antibodies (mAbs) are one of the most important types of biopharmaceutics and have proved enormously successful in the treatment of cancers and autoimmune diseases. In this paper, we present a fast, straightforward reversed phase (RP)UHPLC-UV-(HESI/Orbitrap) MS method for the separation and identification of five of the most commonly used mAbs, i.e. bevazizumab (BEV), cetuximab (CTX), infliximab (INF), rituximab (RTX) and trastuzumab (TTZ) in mixtures. The RP mAbs separation was performed in a divinylbenzene-based monolithic column, after statistical design of the experiments with a novel approach for optimizing chromatographic conditions called the heteroscedasticity function. Results led us to split the initial mixture of five mAbs into two mixtures with four mAbs each, one containing RTX and the other TTZ. The method was validated for quantification using the signal from the UV detector and identification by (HESI-Orbitrap)MS. Direct MS characterization of the intact isoform profile of each mAb was also obtained. Advantages and disadvantages of the use of trifluoroacetic acid or formic acid as ion pairing agents for mass spectrometric analysis and chromatographic separation are discussed. Validation was performed using an internal protocol based on well-known international guidelines such as the International Conference on Harmonization (ICH) guideline, the US Food and Drugs Administration (FDA) guideline and the United Stated Pharmacopeia (USP) guideline. Performance parameters such as linearity, accuracy (precision and trueness), detection limits, quantification limits and robustness were evaluated. Robustness was established by studying the total and one-sided effects of four selected variables: column temperature, trifluoroacetic acid content in the mobile phases, initial proportion of eluent B and gradient. The results indicated the suitability of this method for quantifying these five mAbs in mixtures, as well as its robustness, reproducibility and sensitivity.

Validated reverse phase HPLC diode array method for the quantification of intact bevacizumab, infliximab and trastuzumab for long-term stability study.

The aim of the present study was to develop suitable and reliable method for quantification three of the most worldwide used therapeutic monoclonal antibodies (mAbs) -bevazizumab (BVZ), infliximab (INF) and trastuzumab (TTZ)- to be used in long-term stability studies. Reverse phase (RP) was selected by its greater sensibility and reproducibility comparing with other chromatographic modes. Then a high performance liquid chromatography with diode array detection (RP)HPLC/DAD method was checked. Since the three mAbs represent the active ingredient in the medicines in which they are formulated, the selected method was validated for each one in accordance with the International Conference on Harmonization (ICH) guidelines for pharmaceuticals for human use. Then method was validated in terms of linearity, accuracy, precision, (repeatability, intermediate precision) specificity (by forced degradation studies), robustness and system suitability. Spectral peak purity analysis strategy was used to test mAb degradations. Comparative study of the results indicated similar behavior for the three mAbs. Forced degradation studies also provided deep knowledge of these important bio-macromolecules. At last the method was successfully used to quantify BVZ, INF and TTZ in long-term stability studies performed under hospital conditions of use and they showed great stability regarding quantification during the time of the study.

Study of aggregation in therapeutic monoclonal antibodies subjected to stress and long-term stability tests by analyzing size exclusion liquid chromatographic profiles.

Research into stress and stability is essential during the development of therapeutic proteins to ensure quality and safety of the final medicine. Greater knowledge of the effects of stress on aggregation can help avoid undesirable conformational and colloidal instabilities. With this in mind we investigated five marketed therapeutic monoclonal antibodies (mAbs) namely bebacizumab (BVZ), cetuximab (CTX), infliximab (IFX), rituximab (RTX) and tratuzumab (TTZ) in their innovative medicines. These were submitted to different controlled stresses, to freeze/thaw cycles and used for long-term stability studies once the vials were opened. Aggregate formation was tracked by analyzing the mAbs chromatographic profiles by size-exclusion liquid chromatography coupled with diode array detection. Results indicated that the tendency to aggregate depends on the particular stress conditions and on the concentration and nature of the mAb, even though all share similar IgG1-structure. Fragmentation of the mAb produced by the stress was probably due to the rupture of cystines between the two heavy chains. Regarding stability study, BVZ, RTX and TTZ proved to be the most stable when stored at 4 °C and in freeze/thaw cycles with no tendency to form aggregates. INF tends to form aggregates at 0.5 mg/mL, while in CTX, the most unstable, degradation was detected.