Accelerated CMC workflows to enable speed to clinic in the COVID-19 era: A multi-company view from the biopharmaceutical industry.

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation.

Applying intensified design of experiments to mammalian cell culture processes.

The analysis of data collected using design of experiments (DoE) is the current gold standard to determine the influence of input parameters and their interactions on process performance and product quality. In early development, knowledge on the bioprocess of a new product is limited. Many input parameters need to be investigated for a thorough investigation.

Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing.

Genetically modified CHO cell lines are traditionally used for the production of biopharmaceuticals. However, an in-depth molecular understanding of the mechanism and exact position of transgene integration into the genome of pharmaceutical manufacturing cell lines is still scarce. Next-generation sequencing (NGS) holds great promise for strongly facilitating the understanding of CHO cell factories, as it has matured to a powerful and affordable technology for cellular genotype analysis.

Cysteine in cell culture media induces acidic IgG1 species by disrupting the disulfide bond network.

A high degree of charge heterogeneity is an unfavorable phenomenon commonly observed for therapeutic monoclonal antibodies (mAbs). Removal of these impurities during manufacturing often comes at the cost of impaired step yields. A wide spectrum of posttranslational and chemical modifications is known to modify mAb charge.

Pre-stage perfusion and ultra-high seeding cell density in CHO fed-batch culture: a case study for process intensification guided by systems biotechnology.

Process intensification strategies are needed in the field of therapeutic protein production for higher productivities, lower cost of goods and improved facility utilization. This work describes an intensification approach, which connects a tangential-flow-filtration (TFF) based pre-stage perfusion process with a concentrated fed-batch production culture inoculated with an ultra-high seeding density (uHSD). This strategy shifted biomass production towards the pre-stage, reaching up to 45 × 10 cells/mL in perfusion mode.

Reduction of IL-2 fragmentation during manufacturing of a novel immunocytokine by DoE process optimization.

Interleukin-2 (IL-2) is a potent molecule in cancer therapy. Clinical application, however, is limited due to its strong side effects during the treatment. We developed an IL-2 variant (IL-2v) immunocytokine to circumvent the drawbacks of the current IL-2 therapy.

Visualisation of intracellular production bottlenecks in suspension-adapted CHO cells producing complex biopharmaceuticals using fluorescence microscopy.

With the advance of complex biological formats such as bispecific antibodies or fusion proteins, mammalian expression systems often show low performance. Described determining factors may be accumulation or haltering of heterologous proteins within the different cellular compartments disturbing transport or secretion. In case of the investigated bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cell line neither impaired transcription nor decreased translation processes were identified and thus satisfactorily explained its low production capacity.

Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines.

We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs.

A single-step FACS sorting strategy in conjunction with fluorescent vital dye imaging efficiently assures clonality of biopharmaceutical production cell lines.

The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation.

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g.

Molecular polygamy: The promiscuity of l-phenylalanyl-tRNA-synthetase triggers misincorporation of meta- and ortho-tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells.

In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process.

Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy.

Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation.

Modulation of mesenchymal stromal cell characteristics by microcarrier culture in bioreactors.

Mesenchymal stromal cells (MSCs) are promising candidates for cell therapy. Their therapeutic use requires extensive expansion to obtain a sufficiently high number of cells for clinical applications. State-of-the-art expansion systems, that is, primarily culture flask-based systems, are limited regarding scale-up, automation, and reproducibility.

Low level sequence variant analysis of recombinant proteins: an optimized approach.

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage.

Essential CDK1-inhibitory role for separase during meiosis I in vertebrate oocytes.

Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms, but in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) on liberation from the inhibitory protein, securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar-body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity.

Anaphase topsy-turvy: Cdk1 a securin, separase a CKI.

Chromosome segregation in mitosis and meiosis is triggered by activation of a large protease, separase. While it has been known for some time that the anaphase inhibitor securin regulates separase activity recent work shows that this is only half the story. In vertebrates Cdk1-dependent inhibition of separase represents a second, securin-independent branch of anaphase regulation.

Mutual inhibition of separase and Cdk1 by two-step complex formation.

Stable maintenance of genetic information requires chromosome segregation to occur with high accuracy. Anaphase is triggered when ring-shaped cohesin is cleaved by separase, a protease regulated by association with its inhibitor securin. Dispensability of vertebrate securin strongly suggests additional means of separase regulation.

Rephrasing anaphase: separase FEARs shugoshin.

Cleavage of the ring-like cohesin complex by separase triggers segregation of sister chromatids in anaphase. This simplistic model has recently been extended by exciting discoveries on three levels: regulation of anaphase by posttranslational modifications and the cohesin protector shugoshin; non-proteolytic roles of separase; and cohesin-independent linkage of sister chromatids.