Generation and characterization of a HEK293 cell line optimized for recombinant adeno-associated virus production

Cell lines producing biopharmaceuticals with high yield and high quality in a regulatory compliant environment are a prerequisite for cost effective bioproductions. The development of these production cell lines often includes screening strategies combined with gene amplification and limited dilution experiments, a time consuming process. Especially gene amplification tends to interfere with clonal stability. Limited dilution, especially using a serum-free culture environment is prone to failure and low clone yields. We present here the SEFEX platform technology for the development of non-amplified high yield production cell lines. The strategy is based on a regulatory compliant method for transfection and single cell cloning using a proprietary, fully tested, CHO-K1 host cell line adapted to chemically defined medium. Fast track cell lines were generated by selection of single cell derived clones within 2.5 months after transfection. 1.0 g/L product concentration were achieved after two rounds of process development using these cell lines. Optimized cell lines were developed based on fast track cell lines employing a second transfection. These cell lines were capable for production of 2.6 g/L during an early phase of process development. Cell line development with the SEFEX technology comprises steps carried out entirely under serum-free conditions including transfection, selection, and single cell cloning. Fast track cell lines were developed within 2.5 months by transfection and selection of stably transfected cells followed by single cell cloning. During single cell cloning, semi-automated photo documentation at the single cell level is used to assure and document clonality in a regulatory compliant manner. Table ​Table11 summarizes cell specific productivity and product concentration in fed-batch processes. More than 1.0 g/L were achieved in a GMP process at 300 L scale after two steps of process development followed by scale-up development. A similar scale-up strategy including GMP production was summarized by Landauer et al. in this issue [1]. Nevertheless, production of proteins that are difficult to express like, IgG C in Table ​Table1,1, suffered from low productivity and low yield. Optimized cell lines, which were obtained by a second serial transfection of the antibody expression vector including regulatory sequences, were suitable to deliver high rates of antibody production. The productivity of IgG C was improved 6-fold to18 pg/c/d. This allowed production of the difficult to express protein at a product concentration of 1.1 g/L in fed-batch mode (Table ​(Table1).1). Other examples of optimized cell lines provided product concentrations of 1.7 g/L without any optimization during cell line development (IgG A, Table ​Table1),1), or 2.6 g/L after two steps of upstream process development at 1 L bioreactor scale (IgG B, Table ​Table1,1, Figure ​Figure11). Table 1 Cell specific productivity and product concentration obtained with fast track cell lines and optimized cell lines. Figure 1 Fed-batch experiments for production of IgG B. The fast-track cell line (left hand graph) was cultivated at 10 L scale after two steps of process development and scale up using chemically defined basal and feed medium. The optimized cell line (right hand ... Figure ​Figure11 shows growth curves of the fast-track and optimized cell line producing IgG B. Optimization of maximum viable cell density was not addressed during upstream process development so far. This optimization is currently ongoing and leaves room for further improvements. Specific productivity of the optimized cell lines varied between 18 and 36 pg/c/d, which was equivalent to a two- to six-fold improvement compared to the fast track cell lines. Productivity was suitable for production of high volume products. Scale-up to 300 L stirred tank and 1000 L wave bioreactor for production of clinical phase II drug product was shown for production cell lines generated with the SEFEX platform technology. The SEFEX platform technology for the development of non-amplified high yield production cell lines is based on a regulatory compliant, proprietary CHO-K1 host cell line adapted to chemically defined medium. Fast track cell lines, which were available within 2.5 months produced 1.0 g/L product. Optimized cell lines, which were developed based on fast track cell lines, were capable for production of 2.6 g/L in early phase of process development. Using the SEFEX platform technology, all development steps were carried out under entirely serum-free or chemically defined media conditions including transfection, selection, and single cell cloning. Clonality was assured and documented in a regulatory compliant manner using photo documentation of single cells in cell cloning experiments. The serial transfection cell line development strategy described here provides the possibility to develop production cell lines meeting industrial demands employing simplest process development procedures within minimized time frames.

Researchers need to identify the nephrotoxic properties of medicinal products both during preclinical development and when exploring options to optimise pharmacotherapy. The main challenge is to find an experimental model for assessing drug-induced nephrotoxicity that reflects in vivo conditions as closely as possible.The aim of the study was to compare the susceptibility of HEK293 and RPTEC cell lines used as experimental models for assessing the nephrotoxicity of cefuroxime and cefepime.Materials and methods. The study investigated HEK293 and RPTEC cell lines cultured on plates with 0.4 µm pore membrane inserts. The cell lines were incubated for 3 days with cefuroxime and cefepime (cephalosporins excreted primarily by the kidneys). The medicinal products were added to the basal part of the well at concentrations of 50 and 150 µg/mL (cefuroxime) or 30 and 120 µg/mL (cefepime) twice a day. After incubating the cells with cefuroxime and cefepime for 24, 48, and 72 hours, the authors determined the expression levels of the SLC22A6 and SLC22A8 genes encoding organic anion transporters by a reverse transcription polymerase chain reaction. The authors considered caspase 3 and caspase 7 activation indicative of the nephrotoxic effect of cephalosporins; they evaluated this indicator by a fluorometric assay after 24, 48, and 72 hours of incubation.Results. According to the study, the expression of the SLC22A6 and SLC22A8 genes decreased with cephalosporin transport in both cell lines. The decrease occurred in the RPTEC cell line earlier than in the HEK293 cell line. The authors observed caspase 3 and caspase 7 activation only in the RPTEC cell line after incubation with cefuroxime and cefepime at low concentrations (50 and 30 µg/mL, respectively) for 72 hours and at high concentrations (150 and 120 µg/mL, respectively) for 24 hours.Conclusions. The RPTEC cell line exhibits higher susceptibility to cefuroxime and cefepime toxic effects than the HEK293 cell line due to higher transporter gene expression. Higher cephalosporin concentrations accelerate caspase 3 and caspase 7 activation in the RPTEC cell line. The experimental model based on the RPTEC cell line is a promising tool for the analysis of the nephrotoxic properties of a wide range of medicinal products.

Experimental validation of candidate schizophrenia gene CALN1 as a target for microRNA-137

Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long-standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a, Lig4, Palb2, Pari, and Xrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO-DG44 cell lines. Notably, two genes, Lig4 and Xrcc6, showed higher expression in unstable CHO-DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long-term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

Introduction and objective. Alnus sieboldiana (Betulaceae) is a warm temperate tree distributed in highland areas along the Pacific shores of central Japan. The aim of the study was to evaluate the cytotoxic activity of fractions obtained from n-hexane extract from male flowers of Alnus sieboldiana towards VERO and HEK293 cell lines. Materials and methods. Cytotoxicity was estimated using the MTT method. The following cell cultures were used: Vero (ECACC No. 84113001) and HEK293 (ATCC No. CRL-1573). Results. The results indicate that purified n-hexane fraction (F1) showed lowest cytotoxicity among tested fractions, both towards VERO and HEK293 cell lines, with EC 50 values of 145 and 154 µg/ml, respectively. The highest cytotoxicity (EC 50 =23, EC 5 =10 µg/ml) was observed for fraction F3 on HEK293 cell line. Fractions F2, F3 and F4 showed higher cytotoxicity on HEK293 than on VERO cell line with EC 50 values of 46, 23, 40 and 73, 44, 48 µg/ml, respectively. Fractions F3 and F4 showed similar toxicity on VERO cell line (EC 50 =44, EC 5 =35 µg/ml and EC 50 =48, EC 5 =31 µg/ml, respectively), whereas F3 was significantly more toxic than F4 on HEK293 cell line (EC 50 =23, EC 5 =10 µg/ml and EC 50 =40, EC 5 =25 µg/ml, respectively). Conclusions. The human HEK293 cell line was more sensitive to tested fractions with the exception of F1. Studies on cytotoxicity of A. sieboldiana flowers on VERO and HEK293 cell lines may prove to be useful in the assessment of non-cytotoxic concentration of fractions that will be used during future research on the biological activity of this plant. EtOAc fraction (F3) showed potent cytotoxic activity, especially on HEK293, and therefore should be further analyzed using appropriate cell lines for potential anticancer properties.

Background: Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of paediatric cancer deaths. Multiple genetic abnormalities have been identified as prognostically significant in neuroblastoma patients. Optical genome mapping (OGM) is a novel cytogenetic technique used to detect structural variants, which has not previously been tested in neuroblastoma. We used OGM to identify copy number and structural variants (SVs) in neuroblastoma which may have been missed by standard cytogenetic techniques. Methods: Five neuroblastoma cell lines (SH-SY5Y, NBLW, GI-ME-N, NB1691 and SK-N-BE2(C)) and two neuroblastoma tumours were analysed using OGM with the Bionano Saphyr® instrument. The results were analysed using Bionano Access software and compared to previous genetic analyses including G-band karyotyping, FISH (fluorescent in situ hybridisation), single-nucleotide polymorphism (SNP) array and RNA fusion panels for cell lines, and SNP arrays and whole genome sequencing (WGS) for tumours. Results: OGM detected copy number abnormalities found using previous methods and provided estimates for absolute copy numbers of amplified genes. OGM identified novel SVs, including fusion genes in two cell lines of potential clinical significance. Conclusions: OGM can reliably detect clinically significant structural and copy number variations in a single test. OGM may prove to be more time- and cost-effective than current standard cytogenetic techniques for neuroblastoma.

We have leveraged long-read and spatial sequencing modalities to identify patient-specific oncogenic alterations for treatment of pediatric osteosarcomas. Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults, with over 500 cases identified each year in the United States. OS is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. These structural variants (SVs) can alter gene expression stoichiometrically, but the functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments affecting transcription. SVs can also form extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), our goal is to detect complex tumor-specific SVs that will inform and refine pediatric OS subtypes, revealing new tumor-specific chemotherapy treatment options. We have generated a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies using whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in OS, we have integrated OGM and WGS data for six cell lines, and OGM, WGS, and HiC data for two. SVs were detected using OGM, HiC, and WGS modalities, and we observed 59-61% concordance between SVs called using OGM and HiC, versus 22-45% when comparing with WGS calls. PDX lines assayed using OGM exhibit between 5,135 and 7,514 SVs each, and translocation numbers ranged from 212 to 1,422. These lines exhibit between dozens of potential ecDNA amplicons, including one amplicon which may explain that line’s specific and exceptionally high expression of YAP1 and BIRC2 transcripts. HiC interaction maps for two PDX lines reveal genomic compartments and TAD boundaries in detail, with 3,713 unique TAD boundaries identified between them using 50kb genomic bins. We observe that 60% of these boundaries are consistent between these lines, and 27% exhibit differences that suggest boundary loss or gain. These changing boundaries are associated with numerous SVs and/or copy number discrepancies, and with genomic regions containing genes with dysregulated RNA transcript levels. Incorporating OGM and HiC modalities adds substantially to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew S. Clugston, Megan Ostrowski, Coco Wu, Leanne Sayles, Stanley G. Leung, Vijay Ramani, E. Alejandro Sweet-Cordero, Marcus R. Breese. Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 214.

Single-cell cloning is a critical process in the generation of recombinant protein-producing mammalian cell lines. This process traditionally requires 10–20% fetal bovine serum (FBS) or other sera. Due to the presence of sera, single-cell cloning is a potential source of contamination from animal viruses and other adventitious agents. In order to address the regulatory needs of the biopharmaceutical industry, we have developed an Animal Component-Free (AF) medium designed for single-cell cloning of Chinese Hamster Ovary (CHO) cells. We utilized Design of Experiment (DOE) methodology to optimize the levels of six groups of nutrients (amino acids, trace metals, plant-derived hydrolysates, lipids, vitamins and selenium). The optimized formulation was tested with three recombinant CHO cell lines and was shown to generate comparable results, in terms of clonal survival and growth (80% of positive control in average), to the 10% FBS control. The clones generated using the AF cloning medium from three recombinant CHO cell lines were successfully scaled up to spinner or shaker fl ask cultures in Animal Component-Free culture media. The AF cloning process demonstrated improvement of growth and/or productivity. Transfection of a parental CHO K1 cell line and the subsequent selection and cloning processes were also evaluated in this AF cloning medium. Materials and Methods Cell Lines and Media The stock cultures of the test cells lines (parental CHO K1, CHO AP expressing Secreted Alkaline Phosphatase, Recombinant CHO Line 2 expressing recombinant IgG) were maintained in suspension culture in animal-component free media (Sigma-Aldrich C8862 or proprietary formulation). The Basal Medium supplemented with 10% fetal bovine serum (FBS) was the positive control and included for every experiment. The clonal survival and growth results were reported as Wells with Growth (% of Positive Control) from duplicate 96-well plates. All formulations are supplemented with 4mM L-Glutamine unless otherwise specifi ed.

A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids.

BackgroundImproving the treatment of renal cell carcinoma (RCC) will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7), also known as B-FABP and BLBP, which is expressed normally in radial glial cells of the developing central nervous system and cells of the mammary gland. Melanomas, glioblastomas, and several types of carcinomas, including RCC, overexpress FABP7. The abundant expression of FABP7 in primary RCCs compared to certain RCC-derived cell lines may allow the definition of the molecular components of FABP7's regulatory system.ResultsWe determined FABP7 mRNA levels in six RCC cell lines. Two were highly expressed, whereas the other and the embryonic kidney cell line (HEK293) were weakly expressed FABP7 transcripts. Western blot analysis of the cell lines detected strong FABP7 expression only in one RCC cell line. Promoter activity in the RCC cell lines was 3- to 21-fold higher than that of HEK293. Deletion analysis demonstrated that three FABP7 promoter regions contributed to upregulated expression in RCC cell lines, but not in the HEK293 cell. Competition analysis of gel shifts indicated that OCT1, OCT6, and nuclear factor I (NFI) bound to the FABP7 promoter region. Supershift experiments indicated that BRN2 (POU3F2) and NFI bound to the FABP7 promoter region as well. There was an inverse correlation between FABP7 promoter activity and BRN2 mRNA expression. The FABP7-positive cell line's NFI-DNA complex migrated faster than in other cell lines. Levels of NFIA mRNA were higher in the HEK293 cell line than in any of the six RCC cell lines. In contrast, NFIC mRNA expression was lower in the HEK293 cell line than in the six RCC cell lines.ConclusionsThree putative FABP7 promoter regions drive reporter gene expression in RCC cell lines, but not in the HEK293 cell line. BRN2 and NFI may be key factors regulating the expression of FABP7 in certain RCC-derived cell lines.

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

The oxazolone class of compounds is known to exert a profound effect on malignant cell proliferation, tumor angiogenesis and /or on the established neoplastic vasculature. Additionally, these compounds are generally known to have a low tendency to interact with DNA which is not common with most of the conventional cytotoxic agents. Thus, this class of compounds is of particular interest for the discovery and development of patient-friendly anticancer agents. The initial objective of this study was to synthesize and evaluate 2-substituted 4-arylidene- 5(4H)-oxazolones for their potential anticancer properties. A simple, mild and non-hazardous synthetic methodology has been developed for the preparation of 2-substituted 4-arylidene-5(4H)-oxazolones. The methodology involved lemon juice mediated condensation of N-acyl glycine derivatives including hippuric acid with arylaldehydes in PEG-400 under ultrasound irradiation. All the synthesized compounds were screened via an MTT assay for their potential cytotoxic properties in vitro using the cancerous cell lines e.g. K562 (human chronic myeloid leukemia), Colo-205 (human colon carcinoma), and A549 (human lung carcinoma) and a non-cancerous HEK293 (human embryonic kidney) cell line. Compounds 3a, 3c and 3i showed promising growth inhibition against A549 cell line but no significant effects on HEK293 cell line, indicating their selectivity towards cancer cells. Moreover, their IC50 values suggested that all these compounds were comparable to the reference drug doxorubicin indicating their potential against lung cancer. The 4-arylidene-5(4H)-oxazolone framework presented here could be a new template for the design and discovery of potential anticancer agents especially for lung cancer.

Previous work by Morgan and coworkers on radiation-induced genome instability in Chinese hamster ovary (CHO) cell lines showed that unstable LS-12 cells had persistently elevated levels of reactive oxygen species (ROS) that were likely due to dysfunctional mitochondria. To further investigate the correlation between radiation-induced genome instability and dysfunctional mitochondria, we performed quantitative high-throughput mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally unstable CHO cell lines and their stable unirradiated GM10115 parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. The LS-12 cell line, which exhibited the highest level of ROS among the three unstable cell lines, was characterized by eight significantly down-regulated mitochondrial proteins, all associated with the TCA (tricarboxylic acid). Elevated levels of ROS relative to the unirradiated parental control were also statistically significant for the CS-9 cell line. The protein profile of CS-9 revealed five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation. Elevation of ROS in the unstable 115 cell line was nearly as large as that seen in CS-9 cells but was not statistically significant. The mitochondrial protein profile of 115 cells showed significant down-regulation of acetyl-CoA-acetyltransferase, which was also down-regulated in LS-12, and two other proteins with abundances that were significantly different from control levels but were not directly related to either the TCA or oxidative phosphorylation. These results provide further evidence that elevated ROS and mitochondrial dysfunction are associated with radiation-induced genome instability; however, additional work is required to establish a firm mechanistic relationship between these end points.

We have leveraged long-read and spatial sequencing modalities to identify cancer-specific oncogenic alterations for treatment of osteosarcomas. Osteosarcoma is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. The functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments to affect gene expression. SVs can also lead to the formation of extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers yet they remain poorly understood. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), we studied SVs in osteosarcoma with the goal of elucidating their contribution to cancer development. We studied a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies as well as genomically via whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in osteosarcoma, we integrated OGM and WGS data for all 11 cell lines to describe detailed genome-wide SVs including ecDNA formation. PDX lines assayed using OGM exhibited between 106 and 704 translocations each (versus zero detected in germline controls) and we identified dozens of potential ecDNA amplicons, including one that may explain exceptionally high expression of YAP1 and BIRC3 transcripts. Further, we use HiC to determine which ecDNA amplicons appear to be free-floating based on their genome-wide interaction scores, noting significantly greater interaction in between 33% and 67% of ecDNA detected using OGM and break point graph-based methods. We also used HiC to describe the topological environment resulting from genome-wide SVs and observed potential enhancer hijacking events affecting dozens of known oncogenes including MYC and CDK4. HiC interaction maps among six cell lines reveal genomic compartments and TAD boundaries in detail, with 4,167 unique TAD boundaries identified among six cell lines using 50kb genomic bins. Changing TAD boundaries are associated with numerous SVs and/or copy number discrepancies, and are associated with genomic regions that contain genes with dysregulated RNA transcript levels. Overall, OGM and HiC modalities add considerably to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew Scott Clugston. High-resolution mapping of oncogenic structural changes in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2775.

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development.