Screening Clones for Monoclonal Antibody Production Using Droplet Microfluidics Interfaced to Electrospray Ionization Mass Spectrometry.

As one of the most critical steps in process development for protein therapeutics, clone selection and cell culture optimization require a large number of samples to be screened for high titer and desirable molecular profiles. Typical analytical techniques, such as chromatographic approaches, often take minutes per sample which are inefficient for large-scale screenings. Droplet microfluidics coupled to mass spectrometry (MS) represents an attractive approach due to its low volume requirements, high-throughput capabilities, label-free nature, and ability to handle complex mixtures. In this work, we coupled a modified protein cleanup protocol with a droplet-MS workflow for mAb titer screening to guide clone selection. With this droplet approach we achieved a throughput of 0.04 samples/s with an LoD of 0.15 mg/mL and an LoQ of 0.45 mg/mL. To test its performance in a real-world setting, this workflow was applied to a 35-clone screen, where the top 20% producing clones were identified. In addition, we coupled our sample cleanup protocol to a high-resolution MS and compared the glycan profiles of the high titer clones. This work demonstrates that droplet-MS provides a rapid way of clone screening and cell culture optimization based on titer and molecular structure of the expressed proteins. Future work is aimed at increasing the throughput and automation of this droplet-MS technique.

Experimental and computational characterization of mass transfer in high turndown bioreactors.

Single-use bioreactors (SUBs, or disposable bioreactors) are extensively used for the clinical and commercial production of biologics. Despite widespread application, minimal results have been reported utilizing the turndown ratio; an operation mode where the working range of the bioreactor can be expanded to include low fluid volumes. In this work, a systematic investigation into free surface mass transfer and cell growth in high turndown single-use bioreactors is presented. This approach, which combines experimental mass transfer measurements with numerical simulation, deconvolutes the combined effects of headspace mixing and the free surface convective mass transfer on cell growth. Under optimized conditions, mass transfer across the interface alone may be sufficient to satisfy oxygen demands of the cell culture. Within the context of high turndown bioreactors, this finding provides a counterpoint to traditional sparge-based bioreactor operational philosophy. Multiple monoclonal antibody-producing cell lines grown using this high turndown approach showed similar viable cell densities to those cells expanded using a traditional cell bag rocker. Furthermore, cells taken directly from the turndown expansion and placed into production showed identical growth characteristics to traditionally expanded cultures. Taken together, these results suggest that the Xcellerex SUB can be run at a 5:1 working volume as a seed to itself, with no need for system modifications, potentially simplifying preculture operations.

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning.

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

Metabolomic and quality data for early and late passages of an antibody-producing industrial CHO cell line.

In this article, we provide four data sets for an industrial Chinese Hamster Ovary (CHO) cell line producing antibodies during a 14-day bioreactor run. This cell line was selected for further evaluation because of its significant titer loss as the cells were passaged over time. Four conditions that differed in cell bank ages were run for this dataset. Specifically, cells were passaged to passage 12, 21, 25, and 37 and then used in this experiment. Once the run commenced the following datasets were gathered: 1). Glycosylation data for each reactor 2). Size Exclusion Chromatography (SEC) data for the antibodies produced which allowed for the identification of high and low molecular weight species in the samples (N-Glycan and SEC data was taken on day 14 only). 3/4). Metabolites levels measured using Nuclear Magnetic Resonance (NMR) and liquid chromatography-mass spectroscopy (LC-MS) for all reactors over the time course of days 1, 4, 6, 8, 12, and 14. We also provide a graph of the glutamine levels for cells of different ages as an example of the utility of the data. These metabolomics data provide relative amounts for 36 metabolites (NMR) and 109 metabolites (LC-MS) over the 14-day time course. These data were collected in connection with a co-submitted paper [1].

Enabling speed to clinic for monoclonal antibody programs using a pool of clones for IND-enabling toxicity studies.

The importance of speed to clinic for medicines that may address unmet medical needs puts pressure on product development timelines. Historically, both toxicology and first-in-human clinical materials are generated using the same clonal-derived cells to ensure safety and minimize any development risks. However, cell line development with single cell cloning is time consuming, and aggravated by the time needed to screen for a lead clone based on cell line stability and manufacturability. In order to achieve faster timelines, we have used pools of up to six clones for earlier production of drug substance for regulatory filing-enabling toxicology studies, and then the final single clone was selected for production of clinical materials. This approach was enabled by using platform processes across all stages of early development, including expression vectors, host cell lines, media, and production processes. Through comprehensive cell culture and product quality analysis, we demonstrated that the toxicology material was representative of the clinical material for all six monoclonal antibody programs evaluated. Our extensive development experience further confirmed that using a pool of clones for toxicology material generation is a reliable approach to shorten the early development timeline.

Culture of mouse embryonic stem cells.

In this unit standard culture conditions for mouse embryonic stem cells (mESCs) on primary murine embryonic fibroblast (PMEF or MEF) monolayers, culture conditions without MEF for feeder-independent mESCs, and culture conditions in chemically defined media for both feeder-independent mESCs and feeder-dependent mESCs are described. For expansion of an mESC line, it is crucial that cells maintain their undifferentiated state and their self-renewal capacity, and that they remain karyotypically normal, all of which are necessary for successful chimerization of the germ line upon blastocyst injection. Derivation and culture conditions for the original mESCs have been described (notably Robertson, 1987; Smith, 1991; Nagy et al., 2003), however, as there are more and more mESC lines available, it becomes evident that culture conditions are cell-line specific to some extent, and there is a constant demand for culturing details for mESC lines derived from different mouse strains.