Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level.

We describe a mammalian expression construct (SPLICELECT™) that allows the redirection of a proportion of a secreted protein onto the cell surface using alternative splicing: whereas the majority of the RNA is spliced into a transcript encoding a secreted protein, a weak splice donor site yields a secondary transcript encoding, in addition, a C-terminal transmembrane domain. The different sequence elements can be modified in order to modulate the level of cell surface display and of secretion in an independent manner. In this work, we demonstrated that the cell surface display of stable cell lines is correlated with the level of the secreted protein of interest, but also with the level of heterodimerization in the case of a bispecific antibody. It was also shown that this construct may be useful for rapid screening of multiple antibody candidates in binding assays following transient transfection. Thus, the correlation of product quantity and quality of the secreted and of membrane-displayed product in combination with the flexibility of the construct with regards to cell surface display/secretion levels make SPLICELECT™ a valuable tool with many potential applications, not limited to industrial cell line development or antibody engineering.

Analytical assessment of clonal derivation of eukaryotic/CHO cell populations.

Cell populations that are exclusively composed by descendants from a defined single parental progenitor are referred to as "monoclonal", "clonal" or more correctly as "clonally derived cell population". Clonal derivation of cell lines used in the manufacturing of recombinant biologics is a regulatory requirement that aims to ensure a robust process and consistent quality throughout the life cycle of a product. Clonal derivation of cell lines is usually ensured by the process (e.g. two subsequent rounds of limiting dilution). Here we present an approach to analytically assess the probability of clonal derivation of existing cell populations. Using target locus amplification (TLA) followed by next generation sequencing (NGS), unique genetic features can be identified, for example the integration site of the plasmid in the host cell genome or plasmid-plasmid junctions of the plasmids used for expression of recombinant biologics. Whereas a direct assessment of clonal derivation using TLA/NGS data is challenging due to limitations in specificity, confirmed clonally derived populations generated from the cell line population can be analyzed by qPCR for the presence of the unique genetic features identified by TLA/NGS. In the present study, a statistical analysis allowed the demonstration that two independently generated CHO cell lines were clonally derived with an upper 95% confidence interval limit of a potentially present contaminating population of 1.3%.

Identification of chemically diverse, novel inhibitors of 17ß-hydroxysteroid dehydrogenase type 3 and 5 by pharmacophore-based virtual screening.

17ß-Hydroxysteroid dehydrogenase type 3 and 5 (17ß-HSD3 and 17ß-HSD5) catalyze testosterone biosynthesis and thereby constitute therapeutic targets for androgen-related diseases or endocrine-disrupting chemicals. As a fast and efficient tool to identify potential ligands for 17ßHSD3/5, ligand- and structure-based pharmacophore models for both enzymes were developed. The models were evaluated first by in silico screening of commercial compound databases and further experimentally validated by enzymatic efficacy tests of selected virtual hits. Among the 35 tested compounds, 11 novel inhibitors with distinct chemical scaffolds, e.g. sulfonamides and triazoles, and with different selectivity properties were discovered. Thereby, we provide several potential starting points for further 17ß-HSD3 and 17ß-HSD5 inhibitor development. Article from the Special issue on Targeted Inhibitors.