Removal of B. cereus cereulide toxin from monoclonal antibody bioprocess feed via two-step Protein A affinity and multimodal chromatography.

A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Bacillus cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (~1 fmol injected) and limit of quantification 0.16 ng/mL (~3 fmol injected) over a standard curve with >3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process-associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chromatography process. Cereulide contamination is well known and of concern in food processing, however this research may be the first LC-MS quantification of cereulide contamination, and its clearance, in biopharmaceutical mAb processing. The analytical method may also be used to rapidly screen for cereulide contamination in upstream cell culture process streams, prior to downstream product purification. This will allow appropriate measures to be taken to reduce toxin exposure to downstream bioprocess raw materials, consumables and equipment.

Endotoxin inactivation via steam-heat treatment in dilute simethicone emulsions used in biopharmaceutical processes.

Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam-heat treatment was fit to a four-parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature-related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration-related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre-exponential factor was >>10(12) s(-1) suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam-heat treatment decreased endotoxin levels by 1-2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam-heat treatment. The results from this study show that steam-heat treatment is a viable endotoxin control strategy that can be implemented to support large-scale biopharmaceutical manufacturing.

Lifecycle management for recombinant protein production using mammalian cell culture technology.

Product lifecycle management refers to the oversight process and activities carried out to fully realize the commercial potential and value of a product in the marketplace. It is typical for many changes to be introduced to the production processes and testing methods for biopharmaceutical drugs over their lifetime in the commercial marketplace. Technology lifecycle management, as discussed here, refers to the management of the different phases or generations of processes and methods used to make and test the active biopharmaceutical ingredient or drug product, and the adoption of different devices used to present the drug product to patients. The factors to consider when making changes to a commercial biopharmaceutical manufacturing process as part of a technology lifecycle management program are discussed. A case study outlines one approach taken in bringing forward a major process change to a cell culture process for the production of a therapeutic recombinant protein.

Biochemical relapse following radical prostatectomy and miR-200a levels in prostate cancer.

BACKGROUND: Radical prostatectomy cures the majority of men with clinically localized disease, but up to 30% of men relapse with rising serum PSA levels. Stage, Gleason grade, and pre-operative PSA levels are associated with outcome but do not accurately predict which individuals will relapse. MicroRNA (miRNA) levels are altered in cancer and are associated with progression of disease. The miR-200 family has roles in prostate cancer. METHODS: miR-200a levels were measured in 18 radical prostatectomy samples from men who did not relapse and from 18 who did relapse, matched for stage (all T3), grade, and PSA levels. A pair of cancer and normal prostate cell lines derived from the same radical prostatectomy specimen were transfected with miR-200a to determine the effects on growth, wound healing, and invasion. RESULTS: Comparing the matched samples, 11 of the relapsers contained lower, 2 higher and 5 similar levels to the non-relapsers. Transient transfection of miR-200a significantly reduced cell proliferation in prostate cancer cell lines but did not affect invasiveness. CONCLUSION: miR-200a overexpression reduced prostate cancer cell growth and may have potential, in combination with other markers, in stratifying prostate cancer patients for more intensive monitoring and therapy.

Large scale microarray profiling and coexpression network analysis of CHO cells identifies transcriptional modules associated with growth and productivity.

Weighted gene coexpression network analysis (WGCNA) was utilised to explore Chinese hamster ovary (CHO) cell transcriptome patterns associated with bioprocess relevant phenotypes. The dataset set used in this study consisted of 295 microarrays from 121 individual CHO cultures producing a range of biologics including monoclonal antibodies, fusion proteins and therapeutic factors; non-producing cell lines were also included. Samples were taken from a wide range of process scales and formats that varied in terms of seeding density, temperature, medium, feed medium, culture duration and product type. Cells were sampled for gene expression analysis at various stages of the culture and bioprocess-relevant characteristics including cell density, growth rate, viability, lactate, ammonium and cell specific productivity (Qp) were determined. WGCNA identified six distinct clusters of co-expressed genes, five of which were found to have associations with bioprocess variables. Two coexpression clusters were found to be associated with culture growth rate (1 positive and 1 negative). In addition, associations between a further three coexpression modules and Qp were observed (1 positive and 2 negative). Gene set enrichment analysis (GSEA) identified a number of significant biological processes within coexpressed gene clusters including cell cycle, protein secretion and vesicle transport. In summary, the approach presented in this study provides a novel perspective on the CHO cell transcriptome.

Sustained productivity in recombinant Chinese hamster ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype.

BACKGROUND: The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. RESULTS: Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. CONCLUSION: These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

Predicting cell-specific productivity from CHO gene expression.

Improving the rate of recombinant protein production in Chinese hamster ovary (CHO) cells is an important consideration in controlling the cost of biopharmaceuticals. We present the first predictive model of productivity in CHO bioprocess culture based on gene expression profiles. The dataset used to construct the model consisted of transcriptomic data from 70 stationary phase, temperature-shifted CHO production cell line samples, for which the cell-specific productivity had been determined. These samples were utilised to investigate gene expression over a range of high to low monoclonal antibody and fc-fusion-producing CHO cell lines. We utilised a supervised regression algorithm, partial least squares (PLS) incorporating jackknife gene selection, to produce a model of cell-specific productivity (Qp) capable of predicting Qp to within 4.44 pg/cell/day root mean squared error in cross model validation (RMSE(CMV)). The final model, consisting of 287 genes, was capable of accurately predicting Qp in a further panel of 10 additional samples which were incorporated as an independent validation. Several of the genes constituting the model are linked with biological processes relevant to protein metabolism.

Microarray and proteomics expression profiling identifies several candidates, including the valosin-containing protein (VCP), involved in regulating high cellular growth rate in production CHO cell lines.

A high rate of cell growth (micro) leading to rapid accumulation of viable biomass is a desirable phenotype during scale up operations and the early stages of production cultures. In order to identify genes and proteins that contribute to higher growth rates in Chinese hamster ovary (CHO) cells, a combined approach using microarray and proteomic expression profiling analysis was carried out on two matched pairs of CHO production cell lines that displayed either fast or slow growth rates. Statistical analysis of the microarray and proteomic data separately resulted in the identification of 118 gene transcripts and 58 proteins that were differentially expressed between the fast- and slow-growing cells. Overlap comparison of both datasets identified a priority list of 21 candidates associated with a high growth rate phenotype in CHO. Functional analysis (by siRNA) of five of these candidates identified the valosin-containing protein (VCP) as having a substantial impact on CHO cell growth and viability. Knockdown of HSPB1 and ENO1 also had an effect on cell growth (negative and positive, respectively). Further functional validation in CHO using both gene knockdown (siRNA) and overexpression (cDNA) confirmed that altered VCP expression impacted CHO cell proliferation, indicating that VCP and other genes and proteins identified here may play an important role in the regulation of CHO cell growth during log phase culture and are potential candidates for CHO cell line engineering strategies.

Proteomic profiling of CHO cells with enhanced rhBMP-2 productivity following co-expression of PACEsol.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant protein biopharmaceuticals. The purpose of this study was to investigate differences in the proteome of CHO DUKX cells expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) (G5 cells) compared to cells also expressing soluble exogenous paired basic amino acid cleaving enzyme soluble paired basic amino acid cleaving enzyme (PACEsol) (3C9 cells), which has been previously found to improve the post-translational processing of the mature rhBMP-2 dimer. PACEsol co-expression was also associated with a significant increase (almost four-fold) in cellular productivity of rhBMP-2 protein. Differential proteomic expression profiling using 2-D DIGE and MALDI-TOF MS was performed to compare 3C9 and G5 cells, and revealed a list of 60 proteins that showed differential expression (up/downregulated), with a variety of different cellular functions. A substantial number of these altered proteins were found to have chaperone activity, involved with protein folding, assembly and secretion, as well as a number of proteins involved in protein translation. These results support the use of proteomic profiling as a valuable tool towards understanding the biology of bioprocess cultures.

Feasibility and relevance of global expression profiling of gene transcripts in serum from breast cancer patients using whole genome microarrays and quantitative RT-PCR.

BACKGROUND: Previous studies, by ourselves and others, have indicated that gene transcripts are detectable extracellularly. Advancing on this work, in order to investigate the feasibility of analysing global gene expression profiles and so the possibility in the future of identifying panels of circulating mRNA biomarkers that may be diagnostic, prognostic or predictive for cancer, here we performed the first whole genome microarray analysis of human serum. PATIENTS AND METHODS: RNA was isolated from pre-surgery serum and corresponding breast tumour and normal tissue biopsies, and from post-surgery and normal control serum. Specimens were examined using Affymetrix whole genome microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Of the 54,675 mRNAs/variants analysed, approximately 8% and 45% were called Present in serum and breast tissue specimens, respectively. Differentially expressed genes were identified for each group of specimens analysed. Analysis, by qRT-PCR, of 3 selected transcripts further indicated that the nucleic acids detected were mRNA, not DNA. mRNAs are apparently present in serum and their global detection and identification can be successfully achieved using microarray technologies. CONCLUSION: The potential implication of this novel finding is that using microarrays it may be possible to identify a panel of extracellular mRNAs that are diagnostic, prognostic and/or predictive of outcome for cancer patients.

Differential protein expression following low temperature culture of suspension CHO-K1 cells.

BACKGROUND: To ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37 degrees C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood. RESULTS: In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37 degrees C to 31 degrees C in comparison to standard batch culture maintained at 37 degrees C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-alpha), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2). CONCLUSION: These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.

Transcriptional profiling of gene expression changes in a PACE-transfected CHO DUKX cell line secreting high levels of rhBMP-2.

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry for the production of recombinant human proteins including complex polypeptides such as recombinant human bone morphogenic protein 2 (rhBMP-2). Large-scale manufacture of rhBMP-2 has associated production difficulties resulting from incomplete processing of the recombinant human protein due to insufficient endogenous levels of the paired basic amino acid cleaving enzyme (PACE) in CHO. In order to resolve this issue, CHO DUKX cells expressing rhBMP-2 were transfected with the soluble version of human PACE (PACEsol) resulting in improved amino-terminal homogeneity and a fourfold increase in rhBMP-2 productivity. In this article, we present a microarray expression profile analysis comparing the parental lineage to the higher producing subclone co-expressing PACEsol using a proprietary CHO-specific microarray. Using this technology we observed 1,076 significantly different genes in the high-productivity cells co-expressing PACEsol. Following further analysis of the differentially expressed genes, the Unfolded Protein Response (UPR) component of the endoplasmic reticulum stress response pathway was identified as a key candidate for effecting increased productivity in this cell system. Several additional ER- and Golgi-localised proteins were identified which may also contribute to this effect. The results presented here support the use of large-scale microarray expression profiling as a viable and valuable route towards understanding the behaviour of bioprocess cultures in vitro.

Proteomic profiling of secreted proteins from CHO cells using Surface-Enhanced Laser desorption ionization time-of-flight mass spectrometry.

Chinese hamster ovary (CHO) cells are the most commonly used host cell line for the production of recombinant biopharmaceuticals. These biopharmaceuticals are typically secreted from CHO cells and purified from harvested cell culture media. The purpose of this study was to investigate changes in the secreted proteome of CHO cells over the various stages of the growth cycle using Surface Enhanced Laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Conditioned media samples were collected each day over a 6 day growth period from CHO-K1 cells grown in low serum (0.5% FBS) conditions in monolayer culture. Samples were profiled on a number of ProteinChip arrays with different chromatographic surfaces. From this study, 24 proteins were found to be differentially regulated at different phases of the growth cycle in CHO-K1 cells, when profiled on two chromatographic surfaces, Q10 (anionic) and IMAC30 (metal affinity) ProteinChip arrays.

Drug metabolism-related genes as potential biomarkers: analysis of expression in normal and tumour breast tissue.

The complex role of drug metabolism-related enzymes and their possible influence in cancer development, treatment and outcome has not yet been completely elucidated. There is evidence that these enzymes can activate certain environmental procarcinogens to more toxic derivatives and thus a role has been proposed for them in carcinogenesis. The fact that they can also inactivate a number of chemotherapeutic drugs has raised the possibility of these enzymes influencing the sensitivity of tumour cells to anticancer agents. In this report, we analyse the expression of drug metabolism-related genes within a whole genome microarray study of 104 breast cancer and 17 normal breast specimens. Kaplan-Meier survival curves, Chi-squared, and Cox Regression analyses were used to identify associations between expression of gene transcripts and patients' clinicopathological and survival data. Our results show that several of these genes are significantly expressed in both normal and tumour tissue; in many cases, expression is altered in the tumour specimens as compared to normal breast tissue. Moreover, expression of ARNT2 and GST A1 was correlated with prognosis. Kaplan-Meier analysis showed expression of ARNT2 mRNA to correlate significantly with favourable disease outcome for patients, in terms of both their disease-free survival (P = 0.0094) and overall survival (P = 0.0018) times from diagnosis, while detection of GST A1 mRNA correlated with shortened disease-free survival (P = 0.0131) and overall survival (P = 0.0028). Multivariate analysis indicated GST A1 expression to be an independent prognostic factor for overall survival (P = 0.045). Our results suggest a possible use of ARNT2 and GST A1 as prognostic breast cancer biomarkers.

Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells.

This paper describes the first miRNA analysis carried out on hamster cells specifically Chinese hamster ovary (CHO) cells which are the most important cell line for the manufacture of human recombinant biopharmaceutical products. During biphasic culture, an initial phase of rapid cell growth at 37 degrees C is followed by a growth arrest phase induced through reduction of the culture temperature. Growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase. Using miRNA bioarrays generated with probes against human, mouse and rat miRNAs, we have identified 26 differentially expressed miRNAs in CHO-K1 when comparing cells undergoing exponential growth at 37 degrees C to stationary phase cells at 31 degrees C. Five miRNAs were selected for qRT-PCR analysis using specific primer sets to isolate and amplify mature miRNAs. During this analysis, two known growth inhibitory miRNAs, miR-21 and miR-24 were identified as being upregulated during stationary phase growth induced either by temperature shift or during normal batch culture by both bioarray and qRT-PCR. Sequence data confirmed the identity of cgr-miR-21, a novel Cricetulus griseus ortholog of the known miRNA miR-21. This study offers a novel insight into the potential of miRNA regulation of CHO-K1 growth and may provide novel approaches to rational engineering of both cell lines and culture processes to ensure optimal conditions for recombinant protein production.

Proliferation control strategies to improve productivity and survival during CHO based production culture : A summary of recent methods employed and the effects of proliferation control in product secreting CHO cell lines.

Chinese Hamster Ovary cells are the primary system for the production of recombinant proteins for therapeutic use. Protein productivity is directly proportional to viable biomass, viability and culture longevity of the producer cells and a number of approaches have been taken to optimise these parameters. Cell cycle arrest, particularly in G1 phase, typically using reduced temperature cultivation and nutritional control have been used to enhance productivity in production cultures by prolonging the production phase, but the mechanism by which these approaches work is still not fully understood. In this article, we analyse the public literature on proliferation control approaches as they apply to production cell lines with particular reference to what is known about the mechanisms behind each approach.

MicroRNAs: recently discovered key regulators of proliferation and apoptosis in animal cells : Identification of miRNAs regulating growth and survival.

The relatively recent discovery of miRNAs has added a completely new dimension to the study of the regulation of gene expression. The mechanism of action of miRNAs, the conservation between diverse species and the fact that each miRNA can regulate a number of targets and phenotypes clearly indicates the importance of these molecules. In this review the current state of knowledge relating to miRNA expression and gene regulation is presented, outlining the key morphological and biochemical features controlled by miRNAs with particular emphasis on the key phenotypes that impact on cell growth in bioreactors, namely proliferation and apoptosis.

Phenotypic and global gene expression profile changes between low passage and high passage MIN-6 cells.

The long-term potential to routinely use replacement beta cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous beta cell studies, by ourselves and other researchers, have indicated that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies. Long-term culture was found to be associated with many phenotypic changes, including changes in growth rate and cellular morphology, as well as loss of GSIS. Microarray analyses indicate expression of many mRNAs, including many involved in regulated secretion, adhesion and proliferation, to be significantly affected by passaging/ long-term culture. Loss/reduced levels, in high passage cells, of certain transcripts associated with the mature beta cell, together with increased levels of neuron/glia-associated mRNAs, suggest that, with time in culture, MIN-6 cells may revert to an early (possibly multi-potential), poorly differentiated, 'precursor-like' cell type. This observation is supported by increased expression of the stem cell marker, alkaline phosphatase.

Investigation of the molecular profile of basal cell carcinoma using whole genome microarrays.

BACKGROUND: Skin cancer accounts for 1/3 of all newly diagnosed cancer. Although seldom fatal, basal cell carcinoma (BCC) is associated with severe disfigurement and morbidity. BCC has a unique interest for researchers, as although it is often locally invasive, it rarely metastasises. This paper, reporting the first whole genome expression microarray analysis of skin cancer, aimed to investigate the molecular profile of BCC in comparison to non-cancerous skin biopsies. RNA from BCC and normal skin specimens was analysed using Affymetrix whole genome microarrays. A Welch t-test was applied to data normalised using dCHIP to identify significant differentially-expressed genes between BCC and normal specimens. Principal component analysis and support vector machine analysis were performed on resulting genelists, Genmapp was used to identify pathways affected, and GOstat aided identification of areas of gene ontology more highly represented on these lists than would be expected by chance. RESULTS: Following normalisation, specimens clustered into groups of BCC specimens and of normal skin specimens. Of the 54,675 gene transcripts/variants analysed, 3,921 were differentially expressed between BCC and normal skin specimens. Of these, 2,108 were significantly up-regulated and 1,813 were statistically significantly down-regulated in BCCs. CONCLUSION: Functional gene sets differentially expressed include those involved in transcription, proliferation, cell motility, apoptosis and metabolism. As expected, members of the Wnt and hedgehog pathways were found to be significantly different between BCC and normal specimens, as were many previously undescribed changes in gene expression between normal and BCC specimens, including basonuclin2 and mrp9. Quantitative-PCR analysis confirmed our microarray results, identifying novel potential biomarkers for BCC.