Targeted sequencing for comprehensive genetic characterization of a recombinant CHO cell line

Next generation sequencing has revolutionized genomics, catalyzing an era of personally-tailored therapeutics with enhanced efficacy and safety profiles. This technology also holds great promise for bioprocess and cell line development. The combination of Targeted Locus Amplification (TLA) and next generation sequencing is an emerging approach for the characterization of transgene integration and genetic stability for recombinant cell lines. TLA [de Vree et al., Nature Biotechnology 32, 1019-1025 (2014)] is based on the crosslinking of physically proximal sequences and enables the targeted complete amplification and sequencing of transgenes and their integration sites with greatly increased sequence coverage and depth. Information about integration regions, Single Nucleotide Variants (SNVs), and structural changes in the transgene sequences can be tracked across different clones, over the course of multiple cell line generations and processes. TLA sequencing was successfully applied for the comprehensive genetic characterization of a recombinant monoclonal antibody-expressing CHO cell line. A single transgene integration region with three genome-transgene breakpoints was identified within the host genome. Evidence of genetic rearrangements including vector amplification, duplication, and inversion was found by mapping genome-transgene breakpoints and specific patterns of transgene vector-vector concatamers. A copy number \u3e20 transgene insertions was calculated, and PCR on both genomic DNA and cDNA verified a subset of vector fusions. Transgene sequencing to a median coverage of 2,388 reads per base pair also determined a limited number of homozygous and heterozygous SNVs. To further investigate the cell line’s genetic “fingerprint,” ~30 research cell bank sub-clones were examined after shake flask and bioreactor expansion. A highly specific and conserved transgene vector signature was identified, signifying stability. The targeted sequencing approach applied here can efficiently provide extensive genetic sequence and linkage insight for complex transgene integration regions of recombinant cell lines, enabling improved strategies for constructing and ensuring high producing and stable cell line platforms

Transcriptomic and proteomic analysis of lycopene-overproducing Escherichia coli strains

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.Systems biology represents a powerful method to describe and manipulate phenotypes of interest by incorporating biological information from various levels of cellular organization. Such an approach is illustrated from a library of both rationally-directed and combinatorial gene knockout strains of E. coli recombinantly producing the small molecule lycopene. Global genomic and proteomic expression changes associated with increased lycopene production of mutant E. coli constructs were discovered using whole-genome DNA microarrays and a novel LC-MS technique, respectively. While most genes and proteins showed few expression changes, key differences were identified, including targets distal to the non-mevalonate and precursorsupplying pathways. Based upon the expression data sets, it was hypothesized that the following may be associated with lycopene overproduction: histidine biosynthesis (hisH); the quinone pool (wrbA); acid resistance (ydeO and gadE); the glyoxylate pathway (iclR); NADPH redox balance (pntB); growth rate reduction; and membrane composition. In the pre-engineered background strain, deleting pntB (~20-25%) and ydeO (~30%) each led to moderately increased production; overexpressing wrbA led to 50-100% more production at 8 hours and 5-15% more production at later time points; deleting iclR caused small production increases (~5-10%); and supplementing media with histidine caused the parental and mutant strains to have similar production.(cont.) From these observations, several themes emerged. First, reduced cellular growth and energy conservation appear to be important tradeoffs for increasing lycopene production. Second, reducing overflow metabolism to acetate and corresponding acid stress as well as providing a gluconeogenic flux to increase lycopene precursors appeared beneficial. Next, NADPH availability and balance seemed to be critical production factors. The sS factor is known to affect lycopene accumulation, and it was observed to have far-reaching effects on both the transcriptomic and proteomic data sets. While expression changes were not strictly additive between the five mutant strains examined in comparison to the pre-engineered background strain, a number of these common factors appear to be responsible for the high lycopeneproduction phenotype. This work serves as an important example of incorporating multiple layers of complementary biological information to define a basis for an observed phenotype, demonstrating a powerful paradigm for realizing production increases via systems metabolic engineering.by Brian E. Mickus.Ph.D

Identification of copper as a cell culture media component causing metabolite depletion and product sequence variants

The level of peptide sequence variants in a biologic drug substance batch is a critical product quality attribute that should be monitored and controlled. These sequence variants are typically caused by DNA single nucleotide variants that arise in cloning and amplification, mistranscription due to unstable vector DNA or cell age/production stresses, or mistranslation via tRNA wobble or mischarging. In this work, a low frequency of monoclonal antibody sequence variants was detected by mass spectrometry in a drug substance batch. The variants were distributed throughout the heavy and light chains at average levels of under 1% per site with no apparent codon bias. No product-coding DNA mutations were detected via deep sequencing data. This pattern of low level, widely-distributed variation strongly suggested a misincorporation mechanism via mischarging of aminoacyl-tRNA, presumably due to amino acid depletion during the process. Copper is a critical cell culture media component that can be modulated in fed-batch processes to induce lactate consumption via its role as a cofactor for mitochondrial function and respiration. However, complete consumption of lactate can also trigger reduced levels of other metabolites required for recombinant protein assembly, which can lead to product sequence variants. To investigate the potential relationship between media copper supplementation and sequence variants, various levels of copper were supplemented into the basal media for fed-batch cultures at the 250 mL bioreactor scale. Mass spectrometry analysis of the partially purified antibody indicated a positive correlation between the amount of copper supplemented and the level of detected sequence variants as well as a mechanism for sequence variant reduction via targeted nutrient feeding. This work has identified a potential mechanism of sequence variant generation related to cell culture media copper levels as well as process alterations to prevent such variation in future batches, highlighting the importance of carefully controlling trace metal levels. Additional studies may be required to validate the potential mechanism