Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning.

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

Retrospective assessment of clonal origin of cell lines.

Cell lines used for the manufacture of recombinant proteins are expected to arise from a single cell as a control strategy to limit variability and ensure consistent protein production. Health authorities require a minimum of two rounds of limiting dilution cloning or its equivalent to meet the requirement of single cell origin. However, many legacy cell lines may not have been generated with process meeting this criteria potentially impeding the path to commercialization. A general monoclonality assessment strategy was developed based on using the site of plasmid integration for a cell's identity. By comparing the identities of subclones from a master cell bank (MCB) to each other and that of the MCB, a probability of monoclonality was established. Two technologies were used for cell identity, Southern blot and a PCR assay based on plasmid-genome junction sequences identified by splinkerette PCR. Southern blot analysis revealed that subclones may have banding patterns that differ from each other and yet indicate monoclonal origin. Splinkerette PCR identifies cellular sequence flanking the point(s) of plasmid integration. The two assays together provide complimentary data for cell identity that enables proper monoclonality assessment and establishes that the three legacy cell lines investigated are all of clonal origin.

Metabolomic and quality data for early and late passages of an antibody-producing industrial CHO cell line.

In this article, we provide four data sets for an industrial Chinese Hamster Ovary (CHO) cell line producing antibodies during a 14-day bioreactor run. This cell line was selected for further evaluation because of its significant titer loss as the cells were passaged over time. Four conditions that differed in cell bank ages were run for this dataset. Specifically, cells were passaged to passage 12, 21, 25, and 37 and then used in this experiment. Once the run commenced the following datasets were gathered: 1). Glycosylation data for each reactor 2). Size Exclusion Chromatography (SEC) data for the antibodies produced which allowed for the identification of high and low molecular weight species in the samples (N-Glycan and SEC data was taken on day 14 only). 3/4). Metabolites levels measured using Nuclear Magnetic Resonance (NMR) and liquid chromatography-mass spectroscopy (LC-MS) for all reactors over the time course of days 1, 4, 6, 8, 12, and 14. We also provide a graph of the glutamine levels for cells of different ages as an example of the utility of the data. These metabolomics data provide relative amounts for 36 metabolites (NMR) and 109 metabolites (LC-MS) over the 14-day time course. These data were collected in connection with a co-submitted paper [1].

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines.

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS-/- CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10-19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500-L and 1000-L bioreactors replicated laboratory results using 5-L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

Codon-Directed Determination of the Biological Causes of Sequence Variants in Therapeutic Proteins.

Recombinant monoclonal antibodies (mAbs) manufactured from immortalized mammalian cell lines are becoming increasingly important as therapies. Ensuring the quality of expressed proteins is critical when developing manufacturing processes. Protein sequence variants (PSVs) are a type of product-related variant in which errors in the protein sequence are present. Detecting PSVs and determining their origins, either by DNA mutation or mRNA mistranslation, is critical. Mutations cannot be remediated without developing new clones, which can be costly and time-consuming. In contrast, mistranslation can usually be mitigated by optimizing cell culture conditions. In this work, we first developed a new method to detect low-abundance PSVs with improved sensitivity. Then, a statistical metric was proposed to determine whether the observed PSVs originate from mutation or mistranslation by characterizing the distribution of PSVs. This method was applied to the evaluation of 50 clones from five mAbs programs, allowing for identification of five mutation and 139 mistranslation PSVs. The presence of even a few mutations demonstrates the necessity of clone screening during process development.

Endotoxin-induced cytokine and chemokine expression in the HIV-1 transgenic rat.

BACKGROUND: Repeated exposure to a low dose of a bacterial endotoxin such as lipopolysaccharide (LPS) causes immune cells to become refractory to a subsequent endotoxin challenge, a phenomenon known as endotoxin tolerance (ET). During ET, there is an imbalance in pro- and anti-inflammatory cytokine and chemokine production, leading to a dysregulated immune response. HIV-1 viral proteins are known to have an adverse effect on the immune system. However, the effects of HIV-1 viral proteins during ET have not been investigated. METHODS: In this study, HIV-1 transgenic (HIV-1Tg) rats and control F344 rats (n = 12 ea) were randomly treated with 2 non-pyrogenic doses of LPS (LL) to induce ET, or saline (SS), followed by a high challenge dose of LPS (LL+L, SS+L) or saline (LL+S, SS+S). The gene expression of 84 cytokines, chemokines, and their receptors in the brain and spleen was examined by relative quantitative PCR using a PCR array, and protein levels in the brain, spleen, and serum of 7 of these 84 genes was determined using an electrochemiluminescent assay. RESULTS: In the spleen, there was an increase in key pro-inflammatory (IL1α, IL-1ß, IFN-γ) and anti-inflammatory (IL-10) cytokines, and inflammatory chemokines (Ccl2, Ccl7, and Ccl9,) in response to LPS in the SS+L and LL+L (ET) groups of both the HIV-1Tg and F344 rats, but was greater in the HIV-1Tg rats than in the F344. In the ET HIV-1Tg and F344 (LL+L) rats in the spleen, the LPS-induced increase in pro-inflammatory cytokines was diminished and that of the anti-inflammatory cytokine was enhanced compared to the SS+L group rats. In the brain, IL-1ß, as well as the Ccl2, Ccl3, and Ccl7 chemokines were increased to a greater extent in the HIV-1Tg rats compared to the F344; whereas Cxcl1, Cxcl10, and Cxcl11 were increased to a greater extent in the F344 rats compared to the HIV-1Tg rats in the LL+L and SS+L groups. CONCLUSION: Our data indicate that the continuous presence of HIV-1 viral proteins can have tissue-dependent effects on endotoxin-induced cytokine and chemokine expression in the ET state.

A role for reactive oxygen species in endotoxin-induced elevation of MOR expression in the nervous and immune systems.

This study examined the mechanism by which exposure to lipopolysaccharide (LPS) alters mu-opioid receptor (MOR) expression in immune and neuronal cells using an in vitro conditioned medium model system. We found that LPS stimulated the intracellular accumulation of reactive oxygen species (ROS) and MOR expression in macrophage-like TPA-HL-60 cells. Conditioned medium from the LPS-stimulated TPA-HL-60 cells increased MOR expression in SH-SY5Y cells, a neuronal cell model, through actions mediated by TNF-α and GM-CSF. These data suggest that the endotoxin, LPS, modulates MOR expression in nervous and immune cells via ROS signaling, and demonstrates the crosstalk that exists within the neuroimmune axis.

Methamphetamine-mediated modulation of MOR expression in the SH-SY5Y neuroblastoma cell line.

Methamphetamine (METH) has been shown to induce oxidative stress in SH-SY5Y cells, a neuroblastic, dopaminergic cell line model. In neuronal cells, oxidation of dopamine by auto-oxidative or enzymatic mechanisms leads to the production of reactive oxygen species (ROS). Neuronal cells treated with METH accumulate dopamine, which can ultimately lead to increased levels of ROS. ROS has been shown to mediate the expression of the mu-opioid receptor (MOR). The goal of this in vitro study was to examine the effects of METH on the accumulation of intracellular ROS in SH-SY5Y cells, which could, in turn, modulate MOR expression. Confocal laser scanning microscopy (CLSM) indicated that METH induced intracellular accumulation of ROS, detected as increased fluorescence of rhodamine 123, in a dose- and time-dependent manner. Moreover, accumulation of ROS preceded METH-induced expression of the MOR, which was attenuated by the free radical chelator, vitamin E. Additionally, increased MOR expression was noted following hydrogen peroxide treatment, indicating a role for ROS in mediating MOR expression. Taken together, our data show that METH's effect on MOR expression is dependent upon sublethal levels of intracellular ROS, which suggests a possible coupling of METH- and opiate-mediated intracellular signaling.

Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay.

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 microg/mL). The results show that mass spectrometry-based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

Escherichia coli acetyl-coenzyme A carboxylase: characterization and development of a high-throughput assay.

Bacterial acetyl-coenzyme A carboxylase (ACCase) is a multicomponent system composed of AccA, AccD, AccC, and AccB (also known as BCCP), which is required for fatty acid biosynthesis. It is essential for cell growth and has been chemically validated as a target for antimicrobial drug discovery. To identify ACCase inhibitors, a simple and robust assay that monitors the overall activity by measuring phosphate production at physiologically relevant concentrations of all protein components was developed. Inorganic phosphate production was demonstrated to directly reflect the coupled activities of AccC and AccA/D with BCCP cycling between the two half-reactions. The K(m) apparent values for ATP, acetyl-coenzyme A, and BCCP were estimated to be 60+/-14 microM, 18+/-4 microM, and 39+/-9 nM, respectively. The stoichiometry between the two half-reactions was measured to be 1:1. Carboxy-biotin produced in the first half-reaction was stable over the time course of the assay. The assay was adapted to a high-throughput screen (HTS) 384-well format using a modified published scintillation proximity method. The optimized HTS assay has acceptable Z' factor values and was validated to report inhibitions of either AccC or AccA/D. The assay is not susceptible to signal quenching due to colored compounds.

Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts.

Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts. A set of 7 actinomycetes cultures containing indolocarbazoles, known nonselective kinase inhibitors, and a control set of 22 nonproducing indolocarbazole cultures were evaluated. Using red-shifted dyes (Cy3B or Cy5), the authors identified active samples with minimal interference up to the extract concentrations that are 3 times nonextracted culture levels. In contrast, a significant number of interferences were observed using either a fluorescein competitive FP assay or a [33P]ATP Flashplate assay. This work demonstrates that one can screen natural product extracts at high concentrations successfully using FP technology with red-shifted dyes.

Isolation and characterization of two new antifungal antibiotics from a basidiomycete.

Two novel antibiotics, Sch 484129 (1) and Sch 484130 (2), were isolated from the fermentation broth of a fungal culture, which was identified as a Basidiomycete. The new antibiotics were obtained by ethyl acetate extraction followed by reversed phase HPLC purification. Structure elucidation of 1 and 2 was accomplished by spectroscopic data analyses. Derivatizations of the major component 1 were performed in order to provide definitive structural information. Both components were identified as glycolipids and displayed antifungal activity against Saccharomyces and Aspergillus strains.