Photodegradation of Rituximab and Critical Evaluation of Its Sensibility to Electromagnetic Radiation.

Rituximab is a monoclonal antibody used in the treatment of lymphoma non-Hodgkin. This mAb is photosensitive as it is administrated to the patient by infusion/perfusion; therefore, photostability is a decisive factor in the efficacy of this biologic. To better understand the photodegradation mechanisms of Rituximab, this biologic was exposed to different irradiance conditions. We show in this study that this mAb photodegrade proportionally to irradiance intensities. The main modifications of Rituximab by irradiance correlate to the increase in aggregates, decrease in its Tm, acidic charge variants, oxidation of the Trp (36) in the heavy chain, and decrease in complement-dependent cytotoxicity (CDC) potency. To understand the relationship between real-life photo-exposition conditions and ICH standardized light tests, a full characterization was set up. Worst photo-stress cases were evaluated, 1 and 2 h under direct sunlight through a window, mimicking the ID65 electromagnetic radiation profile. Our results show that only exposition to direct sun irradiance during 2 h, (≈ 150 kluxes•h), increases critically soluble and insoluble aggregates, diminishing Tm, increasing acidic charge variants, and the photooxidation of the Trp (36) in the heavy chain measured by peptide mapping-RP-UPLC-MS. A decrease in CDC below 80% resulted under this condition, which correlates with physicochemical analyses. While inside light-room exposition (similar to ICH test) and ICH conditions do not have any contribution to the degradation of Rituximab measured by these physicochemical and biological analytical methods. These results indicate that exposition of Rituximab to below ≈ 75 kluxes•h of sun light cannot photodegrade critically this biologic inside of its primary container.

Detection and quantification of leached ethylene glycol in biopharmaceuticals by RP-UHPLC.

Biopharmaceuticals are in direct contact with different plastic materials, which can contribute to process-related impurities. Polyethylene terephthalate glycol (PETG) is used for storage and transportation of biopharmaceuticals and it is synthetized from the poly-condensation reaction between ethylene glycol, 1,4-cyclodimethanol and dimethyl terephthalate. PETG bottles are analyzed for such impurities prior to release; however, the nature of the pharmaceutical matrix can extract impurities, so it is important to measure these contaminants in biopharmaceuticals. This study shows a liquid chromatography method for the quantification of ethylene glycol in PETG materials as an alternative to the standard USP colorimetric method. The method is based on the derivatization of ethylene glycol with benzoyl chloride in a Schotten-Baumann reaction. We present a comprehensive method development and validation. The method allows the detection and quantification of leached and extracted ethylene glycol directly in biopharmaceuticals after years of storage in contact with PETG bottles. Results showed residual ethylene glycol in drug substances to a level of ≈  0.1-0.5 µg/mL exposed during 2-6 years of storage in PETG bottles and ≈ 0.2-0.9 µg/mL in biopharmaceuticals. Graphical abstract Biopharmaceuticals must be free or low concentration for leachables, FR-UHPLC-UV analysis is a precise and accurate analytical method for ethylene glycol measurement. This leachable is commonly present in products in direct contact with PETG plastic.

Nanoparticles for Protein Sensing in Primary Containers: Interaction Analysis and Application.

Silver nanoparticles (AgNPs) are known to interact with proteins, leading to modifications of the plasmonic absorption that can be used to monitor this interaction, entailing a promising application for sensing adsorption of therapeutic proteins in primary containers. First, transmission electron microscopy in combination with plasmonic absorption and light scattering responses were used to characterize AgNPs and protein-AgNP complexes, including its concentration dependence, using two therapeutic molecules as models: a monoclonal antibody (mAb) and a synthetic copolymer (SC). Upon interaction, a protein corona was formed around AgNPs with the consequent shifting and broadening of their characteristic surface plasmon resonance (SPR) band (400 nm) to 410 nm and longer wavelenghts. Additional studies revealed secondary and three-dimensional structure modifications of model proteins upon interaction with AgNPs by circular dichroism and fluorescence techniques, respectively. Based on the modification of the SPR condition of AgNPs upon interaction with proteins, we developed a novel protein-sensing application of AgNPs in primary containers. This strategy was used to conduct a compatibility assessment of model proteins towards five commercially available prefillable glass syringe (PFS) models. mAb- and SC-exposed PFSs showed that 74 and 94% of cases were positive for protein adsorption, respectively. Interestingly, protein adsorption on 15% of total tested PFSs was negligible (below the nanogram level). Our results highlight the need of a case-by-case compatibility assessment of therapeutic proteins and their primary containers. This strategy has the potential to be easily applied on other containers and implemented during early-stage product development by pharmaceutical companies and for routine use during batch release by packaging manufacturers.

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method.

This protocol shows the measurement of the apoptotic activity neutralization of TNFα in a mouse fibroblast cell model (WEHI 164) using an anti-TNFα mAb. In addition, this protocol can be used to evaluate other anti-TNFα molecules, such as fusion proteins. The cellular model employed here is sensitive to TNFα-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Comparability of a three-dimensional structure in biopharmaceuticals using spectroscopic methods.

Protein structure depends on weak interactions and covalent bonds, like disulfide bridges, established according to the environmental conditions. Here, we present the validation of two spectroscopic methodologies for the measurement of free and unoxidized thiols, as an attribute of structural integrity, using 5,5'-dithionitrobenzoic acid (DTNB) and DyLight Maleimide (DLM) as derivatizing agents. These methods were used to compare Rituximab and Etanercept products from different manufacturers. Physicochemical comparability was demonstrated for Rituximab products as DTNB showed no statistical differences under native, denaturing, and denaturing-reducing conditions, with Student's t-test P values of 0.6233, 0.4022, and 0.1475, respectively. While for Etanercept products no statistical differences were observed under native (P = 0.0758) and denaturing conditions (P = 0.2450), denaturing-reducing conditions revealed cysteine contents of 98% and 101%, towards the theoretical value of 58, for the evaluated products from different Etanercept manufacturers. DLM supported equality between Rituximab products under native (P = 0.7499) and denaturing conditions (P = 0.8027), but showed statistical differences among Etanercept products under native conditions (P < 0.001). DLM suggested that Infinitam has fewer exposed thiols than Enbrel, although DTNB method, circular dichroism (CD), fluorescence (TCSPC), and activity (TNF α neutralization) showed no differences. Overall, this data revealed the capabilities and drawbacks of each thiol quantification technique and their correlation with protein structure.