Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events.

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥104 colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 104 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.

Design, synthesis, and In vitro antituberculosis activity of 2(5H)-Furanone derivatives.

A series of 2(5H)-furanone-based compounds were synthesized from commercially available mucohalic acids. From the first-generation compounds, three showed inhibitory activity (10 µg/mL) of at least 35% against Mycobacterium smegmatis mc(2) 155 growth (Bioscreen C system). In screening the active first-generation compounds for growth inhibition against Mycobacterium tuberculosis H37Rv, the most active compound was identified with a minimum inhibitory concentration (MIC99 ) of 8.07 µg/mL (15.8 µM) using BACTEC 460 system. No cross-resistance was observed with some current first-line anti-TB drugs, since it similarly inhibited the growth of multidrug resistant (MDR) clinical isolates. The compound showed a good selectivity for mycobacteria since it did not inhibit the growth of selected Gram-positive and Gram-negative bacteria. It also showed synergistic activity with rifampicin (RIF) and additive activity with isoniazid (INH) and ethambutol (EMB). Additional time-kill studies showed that the compound is bacteriostatic to mycobacteria, but cytotoxic to the Chinese Hamster Ovarian (CHO) cell line. From a second generation library, two compounds showed improved anti-TB activity against M. tuberculosis H37Rv and decreased CHO cell cytotoxicity. The compounds exhibited MIC values of 2.62 µg/mL (5.6 µM) and 3.07 µg/mL (5.6 µM) respectively. The improved cytotoxicity against CHO cell line of the two compounds ranged from IC50 = 38.24 µg/mL to IC50 = 45.58 µg/mL when compared to the most active first-generation compound (IC50 = 1.82 µg/mL). The two second generation leads with selectivity indices (SI) of 14.64 and 14.85 respectively, warrant further development as anti-TB drug candidates. © 2016 IUBMB Life, 68(8):612-620, 2016.

Characterization of the specific interaction between sialoadhesin and sialylated Campylobacter jejuni lipooligosaccharides.

In Campylobacter jejuni-induced Guillain-Barré syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for alpha(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.

Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method.

AIMS: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. METHODS AND RESULTS: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. CONCLUSIONS: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time-to-results from 2-5 times shorter than the traditional testing method. SIGNIFICANCE AND IMPACT OF THE STUDY: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans.

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.

YspM, a newly identified Ysa type III secreted protein of Yersinia enterocolitica.

Yersinia enterocolitica has three type three secretion systems, the flagellar, the plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis of Y. enterocolitica and is required for disease processes in the mouse host. Recent data demonstrate a role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersinia secreted proteins). This work characterizes the discovery of a newly identified Ysa type III secreted protein, YspM. Expression of yspM is regulated by temperature, NaCl concentration, and other known regulators of the ysa system. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain of Y. enterocolitica shows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed in Saccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspM(Y295)) was expressed. Site-directed mutagenesis of the histidine of YspM(Y295) ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s).

Accurate and semi-automated analysis of bacterial association with mammalian cells.

To efficiently and accurately quantify the interactions of bacteria with mammalian cells, a reliable fluorescence microscopy assay was developed. Bacteria were engineered to become rapidly and stably fluorescent using Green Fluorescent Protein (GFP) expressed from an inducible Tet promoter. Upon application of the fluorescent bacteria onto a monolayer, extracellular bacteria could be discriminated from intracellular bacteria by antibody staining and microscopy. All bacteria could be detected by GFP expression. External bacteria stained orange, whereas internalised bacteria did not. Internalised bacteria could thus be discriminated from external bacteria by virtue of being green but not orange fluorescent. Image acquisition and counting of various fluorophore-stained entities were accomplished with a high-content screening platform. This allowed for semi-automated and accurate counting of intracellular and extracellular bacteria.

Detection of the induction of Salmonella enterotoxin gene expression by contact with epithelial cells with RT-PCR.

All strains of Salmonella enterica investigated were found to carry the Salmonella enterotoxin gene (stn) as determined by PCR and hybridization studies. However, when using CHO-K1 cells for testing the toxicity of the strains, not all strains showed a toxic effect (cell elongation) on the cells or did so only at a low level. The cultivation of Salmonella in contact with epithelial cells (IEC-6) led to an increase in the production of toxin. The stn gene expression was detectable with the help of the RT-PCR after 3 h of incubation. The RNA of the strains was isolated, transcribed into cDNA (with MMLV-reverse transcriptase) and amplified using PCR. The PCR products were separated electrophoretically using a polyacrylamide gel and detected by silver staining.

Human corneal epithelial extracellular matrix perlecan serves as a site for Pseudomonas aeruginosa binding.

PURPOSE: Previous data has shown that basement membrane associated perlecan serves as a binding site for Pseudomonas aeruginosa in the wounded mouse cornea. The current study determined whether it also provides a binding site for Pseudomonas aeruginosa in transformed human corneal epithelium. METHODS: Bacterial adherence to transformed human corneal epithelial cells grown in normal or in media containing various inhibitors of glycosaminoglycan synthesis was tested. Bacterial binding was similarly tested in wild-type and in mutant Chinese hamster ovary cell lines naturally deficient in glycosaminoglycan synthesis. Transformed human corneal epithelial extracellular matrix also was tested before and after treatment with anti-proteoglycan monoclonal antibodies or heparinase III before bacterial inoculation. Scanning electron microscopy was used to quantitate adherent bacteria. Intact transformed human corneal epithelial cells or extracellular matrix, the latter either treated or not treated with heparinase III or chondroitin ABC lyase were stained to localize perlecan. RESULTS: Examination of the binding of bacteria to transformed human corneal epithelial cells (normal media vs with inhibitors) and Chinese hamster ovary cell lines suggested that bacterial binding was not associated with the surface of either cell type. In contrast, anti-perlecan antibody, as well as heparinase III decreased the binding of bacteria to corneal extracellular matrix. Fluorescence staining localized perlecan to the extracellular matrix beneath the corneal epithelial cells. CONCLUSIONS: Perlecan localized to the extracellular matrix but not the apical surface of transformed human corneal epithelial cells, provides a binding site for Pseudomonas aeruginosa.

Biological and molecular studies of endogenous retrovirus-like genes in Chinese hamster cell lines.

Two types of endogenous retroviral-like sequences related to type C or Intracisternal A Particle (IAP) genes respectively were detected in the Chinese hamster (CH) genome. Their structure and expression were analysed by virological and molecular approaches in different CH cell lines. Several clones were isolated from a CHO genomic library by screening--in stringent conditions--with type C (Gross-MuLV) or IAP (pMIA1) specific probes. These clones were further characterized by restriction mapping and sequencing. Transcripts of type C and IAP-like retroviral sequences were revealed either by PCR or by Northern blot analysis and hybridization with the respective specific probes. However only a few retroviral-like type C particles and no IA particles were detectable by electron microscopy. Only weak activation of type C viral production by halogenated pyrimidines or other classical viral activation agents was observed. No replication of this virus could be obtained in rodent, canine, simian or human cell lines.

Contamination of genetically engineered CHO-cells by epizootic haemorrhagic disease virus (EHDV).

The characterization of a contaminating virus which was detected in genetically-engineered Chinese hamster ovary (CHO) cells during the production of biologicals is described in the present paper. Under electron microscopy, the contaminating virus had a morphology resembling that of an orbivirus. The relationship was confirmed by nucleic acid analysis which showed a RNA segment pattern characteristic of orbiviruses. With an immunoperoxidase staining of monolayer cells and through sero-neutralization tests the virus was identified as being identical to Epizootic Haemorrhagic Disease Virus (EHDV), isolate 318 (untyped) from Bahrain. Potential sources of the contaminating virus and feasible procedures to avoid adventitous virus infections in cell cultures are discussed.

Isolation and characterization of a mutant Chinese hamster ovary cell line that is resistant to Chlamydia trachomatis infection at a novel step in the attachment process.

Host factors involved in Chlamydia trachomatis pathogenesis were investigated by random chemical mutagenesis of Chinese hamster ovary (CHO-K1) cells followed by selection for clones resistant to chlamydial infection. A clonal mutant cell line, D4.1-3, refractory to infection by the C. trachomatis L2 serovar was isolated. The D4.1-3 cell line appears to be lacking in a previously undescribed temperature-dependent and heparin-resistant binding step that occurs subsequent to engagement of cell surface heparan sulfate by L2 elementary bodies. This novel binding step differentiates the lymphogranuloma venereum (LGV) serovar from other serovars and may contribute the different pathologies associated with LGV and non-LGV strains.

Defective endogenous retrovirus-like sequences and particles of Chinese hamster ovary cells.

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. To investigate the origin and expression of these particles, retrovirus-like sequences which are actively transcribed in CHO cells have been cloned and characterized. Two families of sequences related to intracisternal A-particle (IAP) genomes of mice and Syrian hamsters were identified in cytoplasmic RNA from CHO cells (CHO IAP family I and family II). None of the four clones which were sequenced exhibited intact gag, pol, or env open reading frames. Only IAP family II sequences were present in purified extracellular particles of CHO cells. Several cDNA sequences related to mammalian C-type retrovirus genomes were isolated and cloned from gradient-purified, extracellular particles of recombinant CHO cells. All were homologous to the conserved endonuclease domain of murine leukemia virus. Nucleotide sequence analysis of the largest cDNA revealed multiple interruptions of the endonuclease encoding reading frame providing one possible explanation for the non-infectious nature of the particles observed in CHO cells. Both types of retrovirus-like sequences identified in purified extracellular particles of CHO cells (CHO IAP family II and C-type) were present as conserved, moderately repetitive sequences in DNA of all CHO cell lines examined, as well as in DNA from a Chinese hamster liver. It is therefore likely that the extracellular retrovirus-like particles of CHO cells are the products of endogenous provirus elements present in the germline of Chinese hamsters.

Recent studies on retrovirus-like particles in Chinese hamster ovary cells.

Highly concentrated (4000-7000-fold) culture fluids from CHO cells were analysed for the presence of retrovirus-like activity. Concentrates containing reverse transcriptase activity were detected and further purified by sucrose density gradient centrifugation. Particles banding at 1.13-1.16 g/ml were found to contain nucleic acid sequences and structural proteins related to those found in murine and other retroviruses. Analysis of the endonuclease gene has shown no intact open reading frames. Approximately 100-300 copies of the C-type sequences were present in the genome of CHO cell lines as well as in the DNA extracted from Chinese hamster liver, indicating that these sequences are present in the germ line of the species. Concentrates were analysed for infectivity by direct inoculation and co-cultivation with a series of detector cells. No evidence of infectivity was detected by reverse transcriptase, mink cell S+L- focus assay or by electron microscopic analysis of the inoculated detector cells after at least four passages in culture.

Entry of the lymphogranuloma venereum strain of Chlamydia trachomatis into host cells involves cholesterol-rich membrane domains.

Chlamydiae are bacterial pathogens which develop strictly inside the epithelial cells of their hosts. The mechanism used by chlamydiae to enter cells is not well characterized; however, it is thought to consist of a receptor-mediated process. In addition, the formation of clathrin-coated pits appears to be dispensable for chlamydiae to be internalized by host cells. Clathrin-independent endocytosis has recently been shown to occur through cholesterol-rich lipid microdomains, which are characterized by detergent insolubility. In the present study, we investigated whether these lipid domains play a role in Chlamydia trachomatis serovar L2 internalization by host cells. Our results show that after binding to HeLa cells, chlamydiae are associated with detergent-resistant lipid microdomains (DRMs), which can be isolated by fractionation of infected HeLa cells and flotation on a sucrose gradient. After internalization by HeLa cells, chlamydiae were still found in DRMs. In addition, extraction of plasma membrane cholesterol inhibited infection of HeLa cells by C. trachomatis. Many of the proteins associated with DRMs are glycosylphosphatidylinositol (GPI)-anchored proteins; however, our results could not identify a role for GPI-anchored proteins in the entry process. The same results were obtained for Chlamydia psittaci strain GPIC. We propose that cholesterol-rich domains participate in the entry of chlamydiae into host cells. Chlamydia binding to cholesterol-rich domains may lead to coalescence of the bacterial cells, which could trigger internalization by host cells.

The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates.

Using well-characterized mutant host cell lines, deficient in specific enzymes of energy and nucleotide metabolism, we addressed numerous questions regarding nucleotide metabolism in the obligate intracellular bacterium Chlamydia trachomatis. The results presented indicate that C. trachomatis: (i) does not absolutely depend on mitochondrial generated ATP for survival; (ii) does have a significant draw on host-cell NTP pools but does not have a detrimental effect on the ability of the host cell to maintain its energy charge; (iii) lacks the ability to synthesize purine and pyrimidine nucleotides de novo; (iv) is not capable of interconverting purine nucleotides; and (v) possesses the pyrimidine metabolic-pathway enzymes CTP synthetase and deoxycytidine nucleotide deaminase. In total our results indicate that C. trachomatis is auxotrophic for host-cell ATP, GTP and UTP. In contrast, CTP can be obtained from the host cell or it can be synthesized from UTP by the parasite.

Enterotoxin and cytotoxin production by Salmonella enteritidis strains isolated from gastroenteritis outbreaks.

Seventy-six Salmonella enteritidis, three Salmonella virchow and one Salmonella bradenrup strains were screened for enterotoxigenicity by using the Chinese hamster ovary (CHO), Y1 adrenal, Vero and HeLa cell tests. All the strains gave positive reactions for enterotoxin production, except one, and the relative sensitivity to the toxin exhibited by the different cell lines was evaluated. An enterotoxic activity has been identified in sonicated extracts of Salm. enteritidis. This enterotoxin was purified on Agarose A-5m (Bio-Rad) and Superose 12 HR 10/30 column. The enterotoxic activity was eluted from the Superose column in the first peak. Like Vibrio cholerae toxin CT and Escherichia coli enterotoxin LT, it was blocked by GM1 ganglioside, but at a higher concentration. In addition, a cytotoxic factor has been partially identified. The procedure for isolating the cytotoxin included ammonium sulphate precipitation, size-exclusion chromatography and anion exchange chromatography. This cytotoxin factor caused inhibition of protein synthesis in cultured cells, as determined by flow cytometry and [3H]-leucine incorporation. Flow cytometry analysis also showed an activation of CHO cells when exposed to this cytotoxic factor resulting in a state of active growth. Cytotoxic activity was not blocked by gangliosides.

Effect of enteropathogenic Escherichia coli on adherent properties of Chinese hamster ovary cells.

Enteropathogenic Escherichia coli (EPEC) O111:H2, O119:H6, or O142:H6 caused rapid detachment of Chinese hamster ovary (CHO) cell monolayers within 2 to 4 h of cocultivation. CHO cell detachment was not promoted by nonenteropathogenic E. coli (O125:H4, O126:H27, O157:H7, and O26:H11) and could not be attributed to EPEC production of enterohemolysin or Shiga-like toxins. In contrast, EPEC strains did not promote rapid detachment of Lec1, Lec2, or Lec8 CHO cell monolayers. These CHO cell Lec mutants all express abbreviated glycan sequences on membrane glycoproteins and glycolipids. Although EPEC strains failed to alter the adherent properties of Lec2 cells lacking only terminal sialic acid groups, EPEC adherence to the Lec2 mutant was indistinguishable from that observed with wild-type CHO cells. There was also no significant difference in EPEC-induced actin accumulation or invasion of Lec2 cells. In contrast, EPEC localized adherence to Lec1 and Lec8 mutants, lacking sialyllactosamine (Lec1) or sialic acid and galactose (Lec8) sequences, was reduced by 84 and 93%, respectively. Our results suggest that lactosamine sequences [beta Gal(1-4 or 1-3)beta GlcNAc] not containing sialic acid are sufficient for EPEC adherence, actin accumulation, and invasion of CHO cells. Sialic acid groups, however, may be necessary for EPEC-mediated CHO cell detachment.

Isolation, and virulence profiles, of Aeromonas hydrophila implicated in an outbreak of food poisoning in Sweden.

A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A group of 27 people consumed a typical Swedish food "landgång" which is a type of smörgåsbord containing shrimps with mayonnaise, liver paté, ham, sausage, and legume salad which was purchased from a food store. Twenty-two of the 27 persons became ill within 20-34 hr of consumption of the food and reported the symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One person also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who consumed the left-over food the next day, 2 became ill with similar symptoms. The bacteriological examination of left-over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise, smoked sausage, liver paté and boiled ham. The total number of A. hydrophila in these foods were log 10(6) to log > 10(7) organisms per gram of food sample. A. hydrophila was however, not isolated from legume/mayonnaise salad samples. All the food samples tested showed low numbers of other expected food contaminating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus, fungi and yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to produce beta-hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human intestinal (Henle 407) cells in culture.