In-house CHO HCPs platform: A promising approach for HCPs ELISA monitoring.

A key aspect that must be supervised during the development of recombinant therapeutic products is the potential presence of impurities. Residual host cell proteins (HCPs) are a major class of process-related impurities derived from the host organism that even in trace amount have the potential to affect product quality, safety, and efficacy. Therefore, the product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of making the product as pure as possible. The workhorse of HCP monitoring and quantitation during bioprocessing manufacturing is sandwich ELISA (enzyme-linked immunosorbent assay), which employs polyclonal anti-HCP antibodies for both capture and detection. Commercial ELISA kits developed from Chinese Hamster Ovary (CHO) cell lines are widely applied in early drug development stages (preclinical, phase I, and phase II), but are not specifically designed for a given manufacturer's proprietary cell line, and users do not have control over reagent availability and lot-to-lot consistency. For later development stages, the upstream process-specific method is preferred to guarantee an improved sensitivity and coverage. In agreement with the USP General Chapter 〈1132〉, a platform assay can be used in place of the commercial one through all stages of product development, if already available when product development starts. This proof-of-concept study was carried out to demonstrate the feasibility and the advantages of the development of a proprietary CHO HCPs platform ELISA. Different proprietary mock materials have been characterized and compared by orthogonal bidimensional electrophoresis techniques (SDS-PAGE coupled to SS/WB and 2D DIGE) with the scope of selecting the best antigen-antibody couple for setting up the in-house ELISA. A preliminary evaluation of the in-house method performance has been done in comparison with the commercial assay, demonstrating that the platform method is promising for an accurate and precise CHO HCPs quantification during the early phase product and process development.

Impact of four inorganic impurities - iron, copper, nickel and zinc - on the quality attributes of a Fc-fusion protein upon incubation at different temperatures.

A key aspect that must be supervised during the development of a biotherapeutic is the presence of elemental impurities in the final drug product: they must be quantified as to ensure that their concentrations does not affect patients' safety. Regulatory guidelines such as ICH Q3D provides Permitted Daily Exposure (PDE) limits for those impurities considered having a higher potential safety risk. However, one of the limits of such PDE values is that they account for the safety risk, while alterations of certain Quality Attributes (QA) of a biologic may also take place. In order to understand how certain impurities could affect not only the safety of patients, but also the physicochemical properties of biotherapeutics, here we present a study in which we examined how four commonly observed elemental impurities could impact the QAs of a Fc-fusion protein, under normal storage conditions and after six weeks of incubation at +25 °C and +40 °C. The molecule was indeed treated with increasing concentrations of Ni2+, Cu2+, Zn2+ and Fe3+ and the potential changes in conformation, oxidation, aggregation, and fragmentation were monitored. Our data suggest that keeping the levels of these impurities under the safety threshold limits does not guarantee the product quality. While nickel and zinc slightly altered the physicochemical properties of our Fc-fusion protein, iron and copper appeared to be more harmful for the QAs stability. Indeed, these latter elements might cause significant alterations of the product quality such as to potentially alter its efficacy.

Context-dependent regulation of embryonic stem cell differentiation by mGlu4 metabotropic glutamate receptors.

The mGlu5 receptor is the only metabotropic glutamate receptor subtype expressed by mouse embryonic stem (ES) cells grown under non-differentiating conditions [Cappuccio, I., Spinanti, P. Porcellini, A., Desiderati, F., De Vita, T., Storto, M., Capobianco, L., Battaglia, G., Nicoletti, F., Melchiorri, D., 2005. Endogenous activation of mGlu5 metabotropic glutamate receptors supports self-renewal of cultured mouse embryonic stem cells. Neuropharmacology 1, 196-205]. We now report that ES cells differentiating into embryoid bodies (EBs) progressively lose mGlu5 receptors and begin to express mGlu4 receptors at both mRNA and proteinc level. A 4-day treatment of EBs with the mGlu4 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), increased mRNA levels of the mesoderm marker, brachyury and the endoderm marker, H19, and decreased the expression of the transcript for the primitive ectoderm marker, fibroblast-growth factor-5 (FGF-5). These effects were prevented by the mGlu4 receptor antagonists, alpha-methylserine-O-phosphate (MSOP). Plating of EBs for 4 days in vitro in ITSFn medium induced cell differentiation towards a neural lineage, as reflected by the expression of the intermediate filament protein, nestin, and the homeobox protein, Dlx-2. Pharmacological activation of mGlu4 receptors during cell incubation in ITSFn medium increased the expression of both neural markers. Similar results were obtained when neural differentiation was induced by exposure of EBs to retinoic acid. These data suggest that differentiation of cultured ES cells is associated with changes in the expression pattern of mGlu receptors and that activation of mGlu4 receptors affects cell differentiation in a context-dependent manner.