John Mott

Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases†

Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105.

Discovery and Characterization of QPT-1, the Progenitor of a New Class of Bacterial Topoisomerase Inhibitors

ABSTRACT QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the (−)-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic cells, and showed oral efficacy in a murine infection model, all before any medicinal chemistry optimization. Biochemical and genetic characterization showed that the QPT-1 targets the β subunit of bacterial type II topoisomerases via a mechanism of inhibition distinct from the mechanisms of fluoroquinolones and novobiocin. Given these attributes, this compound represents a promising new class of antibacterial agents. The success of this reverse genomics effort demonstrates the utility of exploring strategies that are alternatives to target-based screens in antibacterial drug discovery.

Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by “hard fact” gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO‐K1 and CHO‐S host cells that possess an improved cellular machinery for transient antibody production. The long‐term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re‐transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational. Biotechnol. Bioeng. 2011;108: 386–394.

A study on the temperature dependency and time course of the cold capture antibody secretion assay.

The cold capture assay as described by Brezinsky et al. [Brezinsky, S.C.G., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W., Thill, G., 2003. A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J. Immunol. Methods 277, 141-155] stands out as the most simple of single cell secretion assays which can be used to sort for high productivity in recombinant cell lines. At low temperatures the process of protein release from transport vesicles is assumed to be delayed as both vesicle fusion and product release is slowed, so that secreted proteins can be stained on the cell surface using a fluorescent antibody. Typically, the fluorescent signal obtained correlates to the cell specific production rate of the analysed cell. In the present study we compared staining of human antibody producing CHO cells performed at different temperatures and we observed the fluorescent signal over 24h. We found that the staining temperature did not influence signal intensity. The fluorescent signal was stable for 24h at 4 degrees C, decreased to 80% at room temperature (21 degrees C), while it decreased significantly already after 2h at 37 degrees C. Initially, the fluorescent signal was observed on the cell surface, however, at later stages it was found in compartments in the cytoplasm. Finally we compared differences in signal stability depending on whether the antibody used for staining bound to the light or heavy chain of the product and on whether the fluorescent label was a relatively stable protein (phycoerythrin) or a pH-dependent small molecule (FITC). Our results indicate that the secreted product is trapped by the staining antibody on the cell surface at all temperatures. Subsequently these aggregates are endocytosed by the cells, a process which is slowed down at low temperatures.

Auditioning of CHO Host Cell Lines Using the Artificial Chromosome Expression (ACE) Technology

In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre‐engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO‐S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO‐S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO‐S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO‐S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression. Biotechnol. Bioeng. 2009; 104: 526–539

Resistance mapping and mode of action of a novel class of antibacterial anthranilic acids: evidence for disruption of cell wall biosynthesis

OBJECTIVES The aim of this study was to characterize the mechanism of action of a novel class of bacterial protein synthesis inhibitors identified in a high-throughput coupled transcription-translation assay. METHODS Evaluation of the cross-resistance to antibiotics with known mechanisms of action, resistance mapping and biochemical characterization of a novel class of antibacterial anthranilic acids was performed. RESULTS No cross-resistance to established classes of antibiotics was found. Resistance was mapped to SA1575, an essential, integral membrane protein predicted to be involved in polysaccharide biosynthesis. Biochemical analysis demonstrated the inhibition of cell wall biosynthesis. CONCLUSIONS This novel class of antibacterial anthranilic acids inhibits cell wall biosynthesis. Resistance mapped to SA1575, which may represent a novel target for antibacterial drug discovery.

Elucidation of Essential and Nonessential Genes in the Haemophilus influenzae Rd Cell Wall Biosynthetic Pathway by Targeted Gene Disruption

ABSTRACT Targeted gene disruption by in vitro transposon mutagenesis has been used to identify the genes required for biosynthesis of the Haemophilus influenzae Rd cell wall under standard cultivation conditions. Of the 28 genes known to be associated with the cell wall biosynthetic pathway, 14 were determined to be essential.

Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in chinese hamster ovary cells

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site‐specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary (CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540‐553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed‐batch shake flasks and in a 2‐L fed‐batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed‐batch shake flask process improved titers to 2.5–3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a nonoptimized 2‐L fed‐batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1‐expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO‐dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb‐expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed‐batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.

Development, Validation, and Application of a Fully Integrated Automation System for Screening and Selection of High Yielding Production Cell Lines

The process of screening and selecting cell lines for the production of biopharmaceuticals can be very labor intensive and may involve screening thousands of cells lines in order to select high yielding producers. We have developed an integrated automation system to increase efficiency and throughput for screening and selection during cell line development. The system was designed to automate three sequential assays: (i) high throughput ELISA to identify high producers; (ii) digital imaging of high producers, and (iii) selection and expansion of high producing cell lines.