Bioprocessing Technology Institute

Augmentation therapy is a treatment option available in the market that has been approved by the U.S. Food and Drug Administration (FDA) for alpha-1-antitrypsin (A1AT) deficient patients. The treatment requires weekly injections of purified A1AT for the patients and relies on plasma donor. The demand for A1AT is also high due to its functional role in various diseases. However, scaling up production of purified human plasma A1AT remained costly and challenging. It is therefore of great interest to generate A1AT at larger scale in ensuring a consistent supply to the market. In this paper, we evaluated the stability and productivity of ten Chinese Hamster Ovary (CHO) single cell clones over 12 weeks. This was followed by scaling up the fed-batch production of A1AT with the selected cell clone in a 10L single-use surface aerated orbital shaken bioreactor SB10-X. The cell specific productivity of the two bioreactor runs were at 9.6 and 12 pg/cell/day (pcd) respectively, which were comparable to shake flasks. While the paper focuses on the possibility to scale up A1AT production, process conditions such as feeding regime could be investigated to further prolong the culture longevity and increase productivity.

Since the remarkable breakthrough of COVID-19 mRNA vaccines, lipid nanoparticles (LNPs) have gained substantial attention as the most cutting-edge clinical formulations for mRNA delivery. PEGylated lipid (PEG-lipid) has been regarded as an essential constituent of LNPs that helps to prolong their systemic circulation by preventing particle aggregation in the blood and sequestration by the mononuclear phagocyte system. Herein, we synthesized a series of mRNA-loaded nanoparticles by replacing ALC-0159 (a PEG-lipid used in the Comirnaty formulation) with amphiphilic PEG-polycarbonate diblock copolymers (PC-HNPs). Interestingly, variations of polycarbonate block length and structure significantly influenced mRNA encapsulation efficiency, transfection potency, colloidal stability, and PEG shedding rate of PC-HNPs. In vivo and ex vivo bioluminescence imaging revealed that upon subcutaneous administration in mice, the leading candidate PC3-HNP achieved lymph node accumulation comparable to that of the conventional ALC-0159-based LNP formulation while avoiding undesirable liver accumulation. Our findings may provide valuable information for the construction of next-generation nanocarriers for effective mRNA delivery.

A comprehensive characterization of biotherapeutics, mandated by regulatory authorities, requires analyses of a protein drug at multiple structure levels. Such multilevel characterization can be performed by mass spectrometry (MS), with established conventional MS-based assays of product quality attributes (PQAs) comprising intact protein and subunit middle-up MS with analytes resolved on a C4 column, and bottom-up peptide mapping with analytes resolved on a C18 column. Recent advances in MS have facilitated the increasing use of middle-down analysis, expanding the qualitative analytical capability of MS for protein characterization. Recent studies using less-retentive reversed-phase LC in bottom-up MS also offer an opportunity for streamlining equipment configuration to a single-column LC-MS setup for multilevel characterization of therapeutic proteins. In this study, we developed a robust middle-down LC-MS method on a ZenoTOF 7600 and evaluated a C4 LC-MS setup for the characterization of NISTmAb, RG7221 bispecific antibody (bsAb), and Fc-fusion etanercept by intact protein, subunit middle-up/down, and bottom-up analyses. Successful multilevel characterization of the analytes using C4 LC-MS was demonstrated; notably, high sequence coverage and comprehensive post-translational modification profiling, including the mapping of all 13 O- and 3 N-glycosylation sites on etanercept in a single run, were obtained by bottom-up C4 LC-MS. This is also the first report on middle-down analysis of the major etanercept TNFR and Fc subunit glycoforms. A streamlined single-column LC-MS setup will enable more robust and efficient MS workflows for PQA assessment and simplify the integration of an LC-MS analyzer as a process analytical technology instrument for biopharma applications.

Lipid nanoparticles are a burgeoning technology which has vast potential to improve chimeric antigen receptor (CAR) T cell immunotherapy. This focused review provides an overview of CAR T cell therapy - highlighting its promises, limitations, and challenges - and describes ways in which lipid nanoparticles (LNPs) can be rationally designed to circumvent some of the challenges. Of particular note are antigen presenting cell-mimetic LNPs, which have the potential to streamline the CAR T cell production process by activating T cells and delivering the CAR transgene in a single step. Although the current clinical standard is ex vivo CAR T cell production, in vivo CAR T cell production represents a potentially transformative alternative. Recent innovations in each production method are described, with a particular emphasis on ways in which LNPs may enable in vivo CAR T cell production. The review concludes with a discussion of safety, immunogenicity, scalability, manufacturing, and regulatory factors which will be essential as LNP-based CAR T cell immunotherapies move toward clinical translation.

Potassium ions (K+) released from dying necrotic tumour cells accumulate in the tumour microenvironment (TME) and increase the local K+ concentration to 50mM (high-[K+]e). Here, we demonstrate that high-[K+]e decreases expression of the T-cell receptor subunits CD3ε and CD3ζ and co-stimulatory receptor CD28 and thereby dysregulates intracellular signal transduction cascades. High-[K+]e also alters the metabolic profiles of T-cells, limiting the metabolism of glucose and glutamine, consistent with functional exhaustion. These changes skew T-cell differentiation, favouring Th2 and iTreg subsets that promote tumour growth while restricting antitumour Th1 and Th17 subsets. Similar phenotypes were noted in T-cells present within the necrosis-prone core versus the outer zones of hepatocellular carcinoma (HCC)/colorectal carcinoma (CRC) tumours as analysed by GeoMx digital spatial profiling and flow-cytometry. Our results thus expand the understanding of the contribution of high-[K+]e to the immunosuppressive milieu in the TME.