Real-time monitoring of biopharmaceutical processes is a key necessity for quality by design manufacturing. This talk describes a platform for the integration and modeling of online process data in combination with spectroscopic sensors to predict product quality in real-time. As a proof-of-concept study, the developed model platform was used to predict the time and process dependent evolution of N-linked glycosylation of a monoclonal antibody in small and large scale bioreactors.

As a collaboration between Boehringer Ingelheim Pharma and DataHow AG, we present a generic hybrid modelling approach able to predict process dynamics of CHO cultivation processes and final quality attributes across different products as a function of the process design in the production bioreactor. This novel approach could support development and process characterization activities by capturing common trends in process performance across various projects and thereby improving prediction capabilities and allowing for less experimental effort.

Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established by antibiotic and/or metabolic selection. In this presentation, I will describe a novel post-transcriptional selection technology that utilizes an siRNA cloned upstream of the gene of interest, which is processed to produce functional siRNAs, enabling cell enrichment. This presentation describes the selection principle and compares the productivity, doubling time and stability of clones with those developed by conventional selection methods.

This presentation will discuss the latest reference genome for the community that has chromosome-length scaffold sequences. In addition, we will discuss the use of genome-scale analyses to identify possible safe harbor regions for targeted integration of transgenes.

• Apollo™X is a cell line development platform, incorporating next generation screening technology, enhanced media selection and technical expertise.
• Cell line development timeline reductions have been achieved through the introduction of a novel single cell cloning method which generates high producing clonal cell lines with both high probability and assurance of monoclonality.
• Incorporation of Targeted Locus Amplification (TLA) technology allows the rapid genetic characterization of clonal cell lines

Understanding Critical Quality Attributes (CQAs) and Critical Process Parameters (CPPs) of cell therapy products is essential for scalable, reproducible, and cost-effective manufacturing. Unlike small-molecule drugs or biologics, cells are highly complex systems whose properties can change during ex-vivo manipulation. We describe multi-omics approaches to consider during development of potential cell therapies that can be used to better identify and correlate key attributes to performance and ultimately identify process parameter specifications.

Cell culture medium optimization is the fine tuning of key nutrients to reach a cell line productivity potential. It is an iterative process of spent medium analysis combined with a DOE approach. Presented here is a new automated benchtop analyzer for rapid cell culture medium analysis and optimization. The data provided by this novel at-line tool may accelerate process development analytics by days to weeks with a simple approach offering more frequent visibility into nutrient levels for various applications.

Increasing cell specific productivities (CSPs) is one of the key issues to improve performance of biopharmaceutical production processes using mammalian cells. Searching for CSP boosting additives, we observed 57% CSP and 50% titer rise in IgG-1 producing CHO-DP12 cells after the addition of S‐(5′‐adenosyl)‐L‐methionine (SAM). Detailed analytics revealed the degradation product MTA (5′‐deoxy‐5′‐(methylthio)adenosine) as the key inductor molecule. Cellular responses were analyzed in detail regarding cell cycle, cell volume and metabolic flux patterns.

Continuous optical and dielectric methods of monitoring mammalian cells in bioprocesses allow the detection of metabolic and morphological changes well before the loss of cell viability as determined by dye exclusion methods such as trypan blue. The presentation will evaluate these alternative methods that allow real-time analysis of the state of cells during production and the possibility of intervention in the bioprocess before loss of viability by the cell population.   

Single-use bioreactors are widely used instruments for scaling up bioprocesses. Orbital shaken bioreactors, like the Kuhner SB series, offer many advantages in contrast to alternative technologies like stirred-tank and wave bioreactors. Here we review application data on the relative performance of this technology across scale and highlight some of the key features of the SB series that make it an elegant solution for scaling any mammalian bioprocess.

Amino acid sequence variants in protein therapeutics have the potential to affect product safety and efficacy. In this case study, significant levels of amino acid misincorporation were identified for a mAb produced by CHO cells. Amino acid misincorporation levels were correlated with amino acid depletion and unbalanced amino acid ratios. An iterative approach was taken to optimize the production bioreactor feeding strategy to successfully mitigate amino acid misincorporation.

Advances in automated bioreactor sampling and machine learning for imaged-based cell analysis have significantly improved our ability to quantify and monitor changes in cell health for bioprocessing development. Here we describe leveraging multiple methods of measuring cell health including the Canty cell imager with Flownamics SegFlow autosampler, biocapacitance probes, image-based and flow cytometry, and standard trypan blue-based quantitation to develop more representative models for online or at-line monitoring.

CHO cell line selection represents a painful bottleneck in biotherapeutic development. The Opto™ CLD workflow on the Beacon® system accelerates CLD by integrating cell enrichment, cloning, culture, productivity and quality screening into a single, automated microscale process that generates the highest titer clones in just 5 days. With in-process detection of product aggregation and FDA-accepted monoclonality assurance, Opto CLD can save valuable development time and help de-risk the path to IND.

With the advent of novel genomic editing tools and efficient sequencing techniques, alternative hosts such as eukaryotic microbes have re-emerged as viable expression systems for next generation manufacturing of biologics. Ongoing efforts focused on engineering these organisms have potential to generate a system capable of high productivity, uniform product quality, and short development timelines.

CHO biologics-producing cell lines must demonstrate consistent growth, productivity, and product quality over the course of manufacturing. To characterize features of stable expression, clonally-derived primary production cell lines and their subclones were studied using high-throughput omics technologies. Differential gene expression analysis identified expression patterns related to differences in clonal heterogeneity, stability, aging, and productivity, identifying genetic pathways which may be useful to improve the clone selection process.

Cell engineering is used to enhance manufacturing processes and improve titer/product quality in CHO cultures. Here we present two case studies where we investigated 1) role of intercellular cell adhesion molecule-1 (ICAM-1) in cell-cell aggregate formation in CHO cultures and 2) unexpected expression of insulin degrading enzyme (IDE) by CHO cells and its contribution to insulin degradation in culture media.

CHO cells exhibit substantial instability, thus requiring careful clone selection and stability testing to ensure production hosts maintain essential production phenotypes and critical quality attributes of the product. One mechanism of production instability stems from extensive DNA damage, so we identified across several CHO cell lines, DNA damage repair machinery that itself was damaged or suppressed. Following replacement of damaged alleles, we demonstrated increased stability of the CHO genome and increased production stability. Thus, by repairing the repairers, one can stabilize lead clones used for biotherapeutic manufacturing.