Development and Characterization of the NISTCHO Reference Cell Line

Abstract

Well-characterized reference materials enable successful collaborations within the scientific community by establishing common reagents for benchmarking studies and reducing the barriers to sharing materials and information. Here, we report the development of NISTCHO, a recombinant Chinese hamster ovary cell line expressing a nonoriginator version of the NISTmAb IgG1. We evaluated candidate clonal cell lines in a fed-batch cell culture model to assess growth and productivity of the cell lines and protein quality attributes of the recombinant IgG produced, which demonstrated suitability of multiple candidates. Selection of a preferred candidate was accomplished through sequencing-based analysis of the transgene integration sites, and a base-pair resolution map of the transgene integration site was developed and verified using PCR-based methods. Lastly, a validation study performed by an independent laboratory confirmed the robustness of the preferred candidate, which has been selected for further development as the NISTCHO reference cell line. Together, these results describe the origin of this new reference material and will serve as the foundation for future interlaboratory studies using the NISTCHO cell line.

Keywords: NISTCHO; cell culture; cell line development; monoclonal antibody; reference cell line; transgene integration sites.

FIGURE 1

All candidate clonal cell lines meet initial criteria for growth and productivity assessments in spin tube cell culture models. (A) Cell line development workflow identifying key steps in selection, single cell cloning, and growth, and productivity evaluation. (B) Growth of candidate clonal cell lines in standard fed‐batch process measured by viable cell density (solid lines) and viability (dotted lines, secondary axis). (C) Titer of candidate clonal cell lines in standard fed‐batch process. (D) Cell‐specific productivity of candidate clonal cell lines in standard fed‐batch process. (E) Growth of candidate clonal cell lines in fed‐batch process using alternative feed strategy measured by viable cell density (solid lines) and viability (dotted lines, secondary axis). (F) Titer of candidate clonal cell lines in fed‐batch process using alternative feed strategy. (G) Cell‐specific productivity of candidate clonal cell lines in fed‐batch process using alternative feed strategy. In panels (B)–(G), clones are represented as follows: clone 2, solid blue squares; clone 6, solid red diamonds; clone 13, solid green triangles; clone 15, open pink squares; clone 21, open aqua diamonds; clone 31 (NISTCHO), open purple triangles. Data are reported as the average of duplicate samples with error bars representing one standard deviation. Absent error bars indicate that one of two duplicate samples was terminated due to low viability. (H) Peak volumetric titer, summarized from panels (C) and (F); data are reported as the average of duplicate samples. (I) Assessment of expression stability based on peak volumetric titer, reported as P20 as a percentage of RCB. RCB, research cell bank.

FIGURE 2

Transgene integration profiles identify a preferred candidate for a reference cell line. (A) Map of the vector integration sites observed in the tested NIST CHO clones through long read sequencing (Nanopore). The vector backbones and transgenes are color‐coded (green and red, respectively), and their sizes are depicted to scale. The identified fusion breakpoints are also indicated. Clones 2 and 6 exhibit identical integration patterns, while Clone 31 displays a single vector integration in chromosome 3. (B) Scaffold‐wide view of transgene integration site identified by whole genome sequencing. At the top, label (A) represents the chromosome 3 scaffold from the CriGri‐PICRH‐1.0 genome assembly. Label (B) represents the contig assembled from the RGEN long‐read sequencing method, aligned to chromosome 3. Label (C) indicates reads aligning to the CHO genome assembly in which the mate in the pair aligned to the expression vector. (C) Estimated copy number per reference genome of the NISTCHO expression vector, reported from the light chain sequence (black bar), heavy chain sequence (gray bar), kanamycin marker in the bacterial backbone (black hatched bar), or glutamine synthetase gene derived from the expression vector (gray hatched bar). All copy numbers are reported relative to the single‐copy reference gene Slc35a1. (D) PCR amplification across the transgene‐vector junction on both the 5′ and 3′ end of the transgene integration site, as schematically outlined at the top of the panel. Expected amplicon sizes calculated from the assembled contig are noted in red. (E) Droplet digital PCR quantification of the transgene‐vector junctions. The ratio of the transgene‐vector junction is reported relative to a single‐copy gene (Slc35a1, black bar) or known three‐copy gene (Fbxw2, gray hatched bar). Error bars represent the 95% confidence interval of the copy number ratio, as reported by the BioRad QuantaSoft software.

FIGURE 3

Product quality attributes meet all selection criteria for cNISTmAb‐MPSG produced by NISTCHO in a spin tube cell culture model. In Figures (A)–(C), data are reported as the average of duplicate samples with error bars representing one standard deviation. (A) Assessment of cNISTmAb‐MPSG aggregation levels in material produced by NISTCHO. (B) Relative abundance of N‐glycan forms, reported as percentage of the total quantified. Rare glycoforms with expected abundance < 1% were not quantified or reported. (C) Relative abundance of charge variant species in cNISTmAb‐MPSG produced by NISTCHO in comparison to RM 8671 NISTmAb. The pI of the dominant peak for RM 8671 NISTmAb was used for assignment of acidic, dominant, and basic peaks in all cNISTmAb‐MPSG material evaluated. (D) Quantitative assessment of peptide coverage and posttranslational modification performed by LC‐MS/MS.

FIGURE 4

Performance comparability of NISTCHO using a bioreactor cell culture model is satisfactory during an interlaboratory study. Data are reported as the average of duplicate samples with error bars representing one standard deviation. In all panels, solid line indicates site 1 (Ambr 15) and dotted line indicates site 2 (Ambr 250). (A) Growth and viability profiles for NISTCHO. (B) Product titer profile for cNISTmAb‐MPSG or cNISTmAb‐NMBL produced by NISTCHO. (C) Metabolite profiles for glucose (black) and lactate (red) in NISTCHO cultures. (D) Metabolite profiles for glutamine (black), glutamate (red), and ammonium (green). (E) Metabolite profiles for potassium (black) and calcium (red).

FIGURE 5

The NISTCHO reference cell line was selected based on predetermined criteria. This decision tree reports how candidates were evaluated based on volumetric titer, stability of recombinant protein expression, transgene integration profile, recombinant product quality attributes, and performance in an interlaboratory study.