FcγRIIIA affinity chromatography complements conventional functional characterization of rituximab.

Analytical and functional characterization of batches of biologics/biosimilar products are imperative towards qualifying them for pre-clinical and clinical investigations. Several orthogonal strategies are employed to characterize the functional attributes of these drugs. However, the use of conventional techniques for online monitoring of functional attributes is not feasible. Liquid chromatography is one of the crucial unit operations during the downstream processing of biopharmaceuticals. In this work, we have demonstrated the utility of FcγRIIIA affinity chromatography as an independent quantitative functional characterization tool. FcγRIIIA affinity chromatography aided in sequential elution of Rituximab glycoform mixtures, based on varying levels of galactosylation, and thereby the affinity for the receptor protein. The predominant glycans present in the three Rituximab glycoform mixture peaks were G0F, G1F, and G2F, respectively. Dissociation rate constants were derived from the chromatographic elution profiles by the peak profiling method, for the control and glucose stress conditions. The glucose stress conditions did not result in unfavorable binding kinetics of Rituximab and FcγRIIIA. The dissociation rate constants of the glycoform mixture 2, predominantly consisting of G1F, were similar to the dissociation rate constants obtained by surface plasmon resonance. Moreover, the glycosylation profiles obtained from chromatographic estimation can be corroborated with the ADCC activity. However, the ex vivo ADCC reporter assay indicated that there was an increase in the effector activity with increasing glucose stress. Thus, FcγRIIIA affinity chromatography permitted three independent assessments via a single analysis. Such approaches can be utilized as potential process analytical technology (PAT) tools in the biosimilar development process.

ADCC enhancement: A conundrum or a boon to mAb therapy?

The ability of antibodies to distinctly identify the antigens is an important feature exploited by the scientific community for the treatment of various diseases. The therapeutic action of monoclonal antibodies (mAbs) is mediated along with the cells of the immune system, such as natural killer cells, T cells and macrophages. The two major mechanisms that govern the therapeutic efficacy of mAbs are the antibody dependent cell mediated cytotoxicity (ADCC) and the complement dependent cytotoxicity (CDC). Consequently, much of the research dedicated to improving their action is focussed on enhancing either of these mechanisms. This manuscript focuses on the strategies to enhance ADCC, for providing more efficacious mAb therapeutics. These approaches essentially bring about changes in the elements of ADCC mechanism, such as the effector cell or the antibody itself and thus favour an enhanced therapeutic response. Several technologies of ADCC enhancement have been developed, based on the success of various strategies advanced by the researchers. These technologies show success with a few antibody therapeutics while they do not work with others. This review presents a detailed overview on these strategies and presents perspectives regarding the same.

Characterization of outcomes of amino acid modifications using a combinatorial approach to reveal physical and structural perturbations: A case study using trastuzumab biosimilar.

Biopharmaceuticals, such as monoclonal antibodies, are considered as life-saving drugs for autoimmune diseases, cancer and infectious diseases. However, biotherapeutics tend to undergo chemical degradation during various stages of manufacturing. The conditions of chemical degradation, along with the physical degradation pathways, have a direct influence on the overall stability, safety and efficacy of these therapeutics. While site-specific chemical changes have been well-explored and investigated using various analytical approaches, the resulting conformational and structural changes have not been much studied. Thus, we explored various biophysical techniques for assessing the influence of three representatives forced degradation conditions viz. oxidation, deamidation, and glycation, in a model therapeutic trastuzumab biosimilar. The site-specific modifications caused by these stress conditions were analysed using high resolution mass spectrometry. While their thermodynamic and conformational consequences were investigated by using differential scanning colorimetry (Nano-DSC), circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), and dynamic light scattering (DLS). The investigated stress conditions resulted in reduced thermodynamic stability of mAb, as confirmed using Nano-DSC. Secondary structure analysis performed with CD spectroscopy indicated detectable structural alterations in the beta sheets of stressed samples. DLS and SV-AUC studies demonstrated an enhanced level of aggregation and fragmentation in presence of all stress conditions. Thus, the biophysical analytical toolkits, when used simultaneously, could offer deeper insights into the subtle conformational changes that result from site-specific chemical modifications in mAbs. Hence, these analytical approaches may serve as significant additions to the battery of techniques used for forced degradation analysis of biopharmaceuticals.

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives.

Cryopreservation is a process used for the storage of mammalian cells at a very low temperature, in a state of 'suspended animation.' Highly effective and safe macromolecular cryoprotectants (CPAs) have gained significant attention as they obviate the toxicity of conventional CPAs like dimethyl sulfoxide (DMSO) and reduce the risks involved in the storage of cultures at liquid nitrogen temperatures. These agents provide cryoprotection through multiple mechanisms, involving extracellular and intracellular macromolecular crowding, thereby impacting the biophysical and biochemical dynamics of the freezing medium and the cryopreserved cells. These CPAs vary in their structures and physicochemical properties, which influence their cryoprotective activities. Moreover, the introduction of polymeric crowders in the cryopreservation media enables serum-free storage at low-DMSO concentrations and high-temperature vitrification of frozen cultures (-80 °C). This review highlights the need for macromolecular CPAs and describes their mechanisms of cryopreservation, by elucidating the role of crowding effects. It also classifies the macromolecules based on their chemistry and their structure-activity relationships. Furthermore, this article provides perspectives on the factors that may influence the outcomes of the cell freezing process or may help in designing and evaluating prospective macromolecules. This manuscript also includes case studies about cellular investigations that have been conducted to demonstrate the cryoprotective potential of macromolecular CPAs. Ultimately, this review provides essential directives that will further improve the cell cryopreservation process and may encourage the use of macromolecular CPAs to fortify basic, applied, and translational research.

Fabrication and characterization of starch-TPU based nanofibers for wound healing applications.

Wound dressings have undergone continuous and substantial evolution over time. Modern bandage materials constitute of electrospun biopolymers that enable rapid and effective wound healing due to the high surface area to volume ratio of the electrospun nanofibers and their porous structure. In the present study, nanofibrous bandages, containing a blend of starch-thermoplastic polyurethane (TPU), were developed by using the electrospinning technique. The electrospun nanofibrous mats were subsequently crosslinked with varying concentrations of glutaraldehyde in order to increase their water stability and mechanical properties. The nanofibrous bandages were characterized for their structural properties using SEM, FTIR, TGA, DSC, as well as for their water retention ability, water vapor transmission rate (WVTR), tensile strength and blood clotting efficiency. Cytotoxicity of the bandages was evaluated using human dermal fibroblast cells. Furthermore, the extent of wound healing enabled by the nanofibrous bandage was ascertained using Sprague-Dawley rats. The results revealed that the starch-TPU nanofibrous bandages facilitated enhanced wound-healing, as compared to the traditional dressing material, such as the cotton gauze.

pH dependent aggregation and conformation changes of rituximab using SAXS and its comparison with the standard regulatory approach of biophysical characterization.

Development of biologics and biosimilars involves extensive physical and structural characterization, which underlines the further course of its implementation. These characterization techniques require considerable standardization and are labor intensive. It is therefore, important to have an immediate, independent and affordable characterization strategy that may meet the regulatory guidelines. In this study, we have compared the standard biophysical characterization of an anti-CD 20 antibody with characterization by small angle x ray scattering (SAXS). Aggregation of this mAb was analyzed using standard techniques like size exclusion HPLC, dynamic light scattering and sedimentation velocity - analytical ultracentrifugation, whereas structure analysis was conducted using mass spectrometry, circular dichroism spectroscopy and fluorescence spectroscopy. Our results demonstrated that the inferences about the state of mAb aggregation and its structure deduced using the standard approaches were comparable to the data interpreted using SAXS. The radius of gyration and the P(r) distribution plot obtained using the SAXS scattering data allowed analysis of aggregation and conformation of mAb via a single experiment. Thus, SAXS can be used as an independent technique to complement orthogonal analysis for determining the aggregation profile and structure of mAbs.