Browsing by Author "Hamaker, Nathaniel K."
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Item Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines(Biotechnology and Bioengineering, 2022-05-04) Hamaker, Nathaniel K.; Min, Lie; Lee, Kelvin H.The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industry-wide consortium collaborated to develop a study to characterize age-dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)-producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO-K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis. More than 1500 HCPs were quantified using complementary proteomic techniques, two-dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age-dependent expression were previously reported to be problematic as high-risk and/or difficult-to-remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical.Item Development of site-specific integration strategies and characterization of protein expression instability to improve CHO cell line engineering(University of Delaware, 2022) Hamaker, Nathaniel K.Chinese hamster ovary (CHO) cells, which are widely used for the production of therapeutic proteins, are prone to phenotypic instability. Cell line instability can manifest as productivity decline over time as well as unexpected changes in the amounts of process-related impurities generated. As industry moves toward implementation of longer bioprocesses (e.g., perfusion cell culture systems), it has become increasingly important to understand and address the challenges posed by cell line instability. Accordingly, site-specific integration (SSI) has recently emerged as a powerful cell line engineering technique by allowing transgenes to be targeted to pre-validated genomic loci capable of supporting high and stable expression. Despite the potential benefits of SSI, its implementation in CHO cells has been hindered by remarkably low efficiencies and relatively higher levels of off-target integration. ☐ Here, we developed toolsets for rapidly evaluating genome editing methods that use programmable endonucleases, such as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). First, the SSIGNAL (SSI and genome alteration) reporter system was established to measure both targeted gene disruption and SSI efficiency in CHO cells. The system was used to compare existing strategies for targeted integration that employ different DNA repair pathways to identify the most efficient option to use as a basis for further method development. ☐ Next, a second reporter system called SSIRI was developed with the added ability to quantify random integration (RI) so that SSI methods could be evaluated according to their targeting accuracies (i.e., SSI/RI ratio). The system was leveraged to develop novel methods to dramatically improve SSI efficiency and targeting accuracy as well as to probe the mechanisms that influence transgene integration in CHO cells. When the optimized methods were applied in practice, high-purity SSI cell pools were obtained and cell line development outcomes were improved. Additionally, it became possible to achieve targeted integration at two loci simultaneously. ☐ Finally, we performed an in-depth characterization of cell line instability over time with respect to host cell protein (HCP) and recombinant protein expression for two industrially-relevant CHO cell lines. For continuous processes, which involve longer culture durations, changing concentrations of HCP impurities could be a potential challenge for downstream processing. Two complementary proteomics methods were used to quantify over 1,500 individual HCPs in bioreactor samples. To aid bioprocess improvement, we compiled a list of HCPs that exhibited age-dependent differential expression and have also been previously characterized as problematic. ☐ Collectively, this work provides advanced tools for improved CHO cell line development and useful insights regarding cell line instability at the protein level to inform rational cell line engineering strategies and support biomanufacturing efforts.Item High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors(Biotechnology and Bioengineering, 2023-04-11) Hamaker, Nathaniel K.; Lee, Kelvin H.Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.