Technical evaluation of RNA-Seq and microarray approaches in comparative transcriptomics analysis of CHO cells

RNA-Seq has been replacing microarrays as the primary tool for comparative transcriptomics analysis. However, successful application of RNA-Seq to profile Chinese hamster ovary cells (CHO), the leading industrial cell line for recombinant protein production, was limited, primarily because of the inadequacy of genomic information for CHO cells. A compromised alternative to perform gene expression analysis and pathway or GO enrichment analysis in CHO cells was to map CHO genes to their mouse orthologs. Recent increased availability of CHO genomic references and the KEGG pathway reference for Chinese hamster has enabled direct gene expression analysis in the genome context of CHO cells. This has also enabled evaluation of the comparability of transcriptomics analysis by using different genome references, methods, and platforms. In this study, we applied both microarray and RNA-Seq technologies to profile two CHO cell lines with similar proliferation index but diverse recombinant protein productivity. Molecular signatures such as differentially expressed genes and KEGG pathways were identified to help understand performance differences despite similar cultivation conditions. Our analysis shows that using mouse orthologs for gene expression analysis generates comparable results as using the Chinese hamster genome reference, which provides justification for most previous transcriptomic studies. Additionally, when compared to microarray analysis, RNA-Seq provides superior outcomes due to its wider dynamic gene expression range and higher genome coverage. We also evaluated sample number and sequencing depth, two important parameters in RNA-Seq based comparative transcriptomics, and our results indicated that increasing replicates was more efficient than increasing sequencing depth for increased power and accuracy in differential gene expression analysis. Overall, this study provides multiple practical insights for improved execution of comparative transcriptomics analysis to understand CHO cells at gene expression level

Evaluation of public genome references for RNA-Seq data analysis in Chinese Hamster ovary cells

Recent advances in next-generation sequencing technologies have led to the emergence of RNA-Seq as the preferred transcriptomic tool in the biopharmaceutical industry. However, an important challenge with deploying RNA-Seq to characterize CHO cells is the absence of a common genomic reference for this species. In most published CHO cell transcriptomic studies, RNA-Seq reads are assembled into de novo genomic references which were subsequently used for mapping of the constituent reads. Such an approach makes it difficult to compare results across studies due to the incomplete and non-universal nature of those assemblies. To address this challenge, we evaluated two publicly available genomes and their derived transcriptomes at the NCBI Reference Sequence Database (RefSeq), including CHO-K1 genome (GCF_000223135.1) and Chinese hamster genome (GCF_000419365.1). When applied for a diverse set of 60 RNA-Seq samples, each with approximately 40 million reads, both genomes showed significantly better mapping rates (~75%) compared to their derived transcriptomes (53-63%). Despite similar annotation, gene content, and KEGG pathway coverage level in both genomes, only 69% of overlapping genes between these two genomes had consistent quantification (i.e., read count) across 60 RNA-Seq samples. Examining genes with quantification discrepancies in a genome browser provides an effective avenue to identify targets for potential genome improvement. Two metrics were proposed to assess the genome-specific difference (consistency) and the sample-specific difference (stringency). Genes with low stringency can introduce biases during the identification of differentially expressed genes and pathways. Given that both genomes for CHO cells are still incomplete, we propose utilization of both in RNA-Seq data analyses until a universal reference with refined genome assembly and gene model annotation is generated

A mathematical modeling framework for determining the probability of obtaining a clonally-derived mammalian cell line

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Development of a high throughput CHO cell glycosylation enzyme Mrna expression profiler

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Novel periodic alternating tangential filtration harvest approach provides incresed volumetric productivity

Perfusion cell culture processes provide opportunities to increase product yield through improved cell growth, increased productivity and extended process duration. Most commonly, perfusion cell culture also leads to a continuous harvest operation and collection of harvested cell culture fluid to be processed downstream. As an alternative to continuous harvest, we evaluated instead a periodic harvest approach applied to a non-steady state perfusion cell culture process using alternative tangential flow (ATF). In this ATF perfusion process, product is first accumulated in the bioreactor using ultrafiltration for 15 days with the product then being harvested by microfiltration at end of the process. To further extend the culture time beyond 15 days and maximize productivity, we investigated a sequence of five periodic harvests from a single upstream bioreactor run. The periodic harvests were achieved using an ATF configuration in which ultrafiltration and microfiltration hollow fiber filters were stacked in series (Figure 1). The ultrafiltration hollow fiber retains the product while the microfiltration filter allows product to be collected in the permeate. Permeate was only drawn from the microfiltration filter during the periodic harvest cycles while the permeate was drawn from the ultrafiltration filter during the none harvest cycle periods. This allowed for the accumulation of product in the bioreactor between the periodic harvests. Five harvest cycles were conducted over a 24-day perfusion process. Each harvest cycle was collected for a day with the first harvest cycle starting on day 11. Please click Additional Files below to see the full abstract

Engineering CHO cell lines for the production of biosimilars of murine cell derived reference products

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Enabling next-generation cell line development using continuous perfusion and nanofluidic technologies

The manufacturing process for a biologic begins with establishing a clonally derived, stable production cell line. Generating a highly productive cell line is time and resource intensive and involves screening of a large number of candidates. While miniaturization and automation strategies can reduce resources and increase throughput, they have matured and recent advances have been incremental. With increasing pressure on time to commercialization and the increasing diversity and complexity of therapies in discovery research, there is a need to transform cell line development to accelerate patient access to novel therapies and nanofluidic technology are on potential solution. In this study, we present cell line development data on the Berkeley Lights integrated platform. Cells are manipulated at a single cell level though use of OptoElectronic Positioning (OEP) technology which utilizes projected light patterns to activate photoconductors that gently moves cells. Common cell culture tasks can be programmed though software allowing thousands of cell lines to cultured simultaneously. Cultures can be interrogated for productivity and growth characteristics while on the chip at ~100-fold miniaturization and continuous perfusion of cell culture medium enables effective and robust cell growth and product concentration despite starting from a single cell. Concepts from perfusion culture are also applied to measure productivity and product quality. We demonstrate that commercial production CHO cell lines can be cultured in this nanofluidic environment and show that sub clone isolation, recovery, and selection are achieved with high efficiency. Overall, this technology has the potential to transform cell line development workflows through the replacement of laborious manual processes with nanofluidics and automation, and can ultimately enable the rapid selection of high performing cell lines

Screening cell growth in simulated continuous manufacturing spin tubes determines optimal media conditions for cell lines

While continuous manufacturing (CM) offers significant advantages over batch, fed-batch, and batch perfusion cultures, it is typically more difficult to screen conditions or troubleshoot issues because of the added complexity to the bioreactor system and the long time duration required to receive representative results. For certain screening factors such as medium, the use of spin tubes (50 mL shaken conical-shape vessels) in a simulated CM (sCM) format can be used to approximate the conditions experienced in an instrumented bioreactor. We will discuss how sCM spin tube performance data was used to troubleshoot bioreactor performance while evaluating five cell lines using multiple medium. Specifically we will show how sCM spin tubes can successfully screen medium performance for parameters such as growth, viability, and productivity. The resultant output from the sCM study was then used to perform a confirmation bioreactor run, with significant process improvement that was in line with expected performance. In summary, we show how sCM spin tubes can be used as an effective tool to screen specific inputs such as media for improved bioreactor performance