Cultured Chinese Hamster Ovary Research Articles

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform.

In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.

MTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.

The multidrug transporter MATE1 sequesters OCs within an intracellular compartment that has no influence on OC secretion in renal proximal tubules.

Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OC transporter (OCT)2-mediated uptake from the blood followed by apical multidrug and toxin extruder (MATE)1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H(+) exchanger. As assessed by epifluorescence microscopy, cultured Chinese hamster ovary (CHO) cells that stably expressed human MATE1 accumulated the fluorescent OC N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in human OCT2-expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC [(3)H]1-methyl-4-phenylpyridinium (MPP) from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of V-type H(+)-ATPase, and accumulation of [(3)H]MPP and [(3)H]NBD-MTMA was reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA, suggesting that the influence of BAF was a secondary effect involving inhibition of V-type H(+)-ATPase. The accumulation of [(3)H]MPP by isolated single nonperfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [(3)H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5 μM BAF, suggesting that vesicles loaded with OCs(+) are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion.

Potentially lethal damage repair in drug arrested G2-phase cells after radiation exposure.

Potentially lethal damage (PLD) repair has been defined as that property conferring the ability of cells to recover from DNA damage depending on the postirradiation environment. Using a novel cyclin dependent kinase 1 inhibitor RO-3306 to arrest cells in the G2 phase of the cell cycle, examined PLD repair in G2 in cultured Chinese hamster ovary (CHO) cells. Several CHO-derived DNA repair mutant cell lines were used in this study to elucidate the mechanism of DNA double-strand break repair and to examine PLD repair during the G2 phase of the cell cycle. While arrested in G2 phase, wild-type CHO cells displayed significant PLD repair and improved cell survival compared with cells released immediately from G2 after irradiation. Both the radiation-induced chromosomal aberrations and the delayed entry into mitosis were also reduced by G2-holding PLD recovery. The PLD repair observed in G2 was observed in nonhomologous end-joining (NHEJ) mutant cell lines but absent in homologous recombination mutant cell lines. From the survival curves, G2-NHEJ mutant cell lines were found to be very sensitive to gamma-ray exposure when compared to G2/homologous recombination mutant cell lines. Our findings suggest that after exposure to ionizing radiation during G2, NHEJ is responsible for the majority of non-PLD repair, and conversely, that the homologous recombination is responsible for PLD repair in G2.

Characterization and downstream mannose phosphorylation of human recombinant α‐L‐iduronidase produced in Arabidopsis complex glycan‐deficient (cgl) seeds

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (Aldurazyme™). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells—UDP-GlcNAc:lysosomal enzyme N−acetylglucosamine (GlcNAc)−1−phosphotransferase—and GlcNAc−1−phosphodiester α−N−acetylglucosaminidase (the ‘uncovering enzyme’). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.

Identification of two antagonists of the scavenger receptor CD36 using a high-throughput screening model

CD36, a class B scavenger receptor, is an integral membrane protein that mediates the endocytosis of modified lipoproteins. The functions of CD36 are complex and have been associated with atherosclerosis. In the current study, we developed a high-throughput screening (HTS) assay to identify small molecule antagonists by expressing human CD36 using a Bac-to-Bac baculovirus expression system in Spodoptera frugiperda (Sf9) cells. Uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI–AcLDL) revealed that the IC 50 values for the CD36 ligands oxidatively modified LDL (Ox-LDL), Ac-LDL, and high-density lipoprotein (HDL) were 0.039, 0.019, and 0.010 μg/ml, respectively. Using the HTS assay, two novel compounds, 2016481B and 2038751B, were found to inhibit DiI–AcLDL uptake in insect cells and exhibited IC 50 values of 17.4 and 23.7 μM, respectively. These two novel compounds also inhibited DiI–AcLDL uptake in cultured Chinese hamster ovary (CHO) cells permanently expressing human CD36. Furthermore, these two compounds inhibited lipid accumulation in RAW 264.7 murine macrophage cells in foam cell assays. This HTS assay represents a potential method for identifying more effective macrophage scavenger receptor antagonists, which may serve as starting points for the development of novel anti-atherosclerotic agents.

3D-Frap of PAGFP Reveals Inhomogeneity in Cytoplasmic Structures between the Major Rod Photoreceptor Compartments

Introduction: Diffusion of signaling proteins is thought to be essential for photoreceptor physiology, allowing, for example, regulation of the photoresponse through dynamic sequestration of key transduction proteins in either of the two major photoreceptor compartments, the inner and outer segments (IS and OS, respectively). The influence of the physical nature of the cytoplasm in these compartments on protein mobility is not known, but is essential for understanding photoreceptor function. We have thus developed a novel approach to quantifying protein mobility in 3D in live photoreceptors.Methods: Transgenic Xenopus laevis expressing the photoactivatable variant of GFP, PAGFP, exclusively in rod photoreceptors were generated using established methods. Live retinal slices were imaged with a custom-built multiphoton/confocal microscope. A source of photoactivated PAGFP was generated within selected compartments using multiphoton excitation at 820nm and its dissipation was monitored with 488nm confocal scanning. Images were compared with the output of a 3D diffusion model to estimate effective radial and axial diffusion coefficients.Results: PAGFP diffusion in the IS was isotropic and faster than in the OS, DIS = 5.2 μm2 s−1. In the OS PAGFP diffusion was anisotropic, with faster radial diffusion, DOS-radial = 3.5 μm2 s−1, and slower axial diffusion, DOS-axial = 0.19 μm2 s−1.Conclusion: PAGFP diffusion in both compartments was substantially retarded relative to aqueous solution, Daq,PAGFP ∼ 90 μm2 s−1, and cultured Chinese hamster ovary (CHO) cell cytoplasm, DCHO,PAGFP ∼ 20 μm2 s−1. Moreover, axial diffusion of PAGFP in the OS was hindered to a larger extent than expected from the geometry of disc membranes that span the compartment orthogonal to the cylinder axis. These results suggest that the photoreceptor cytoplasm possess higher density of cytoskeleton and/or macromolecules.

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells

Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co-expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over-expression of the yeast 'protein disaggregase' Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over-expression of the yeast or mammalian Hsp70/40 chaperones. Over-expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5-fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over-expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over-expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells.

A systematic study of the function of the human β‐globin introns on the expression of the human coagulation factor IX in cultured Chinese hamster ovary cells

Intronic sequences have the potential to improve gene expression in eukaryotes by a variety of mechanisms. In this context, human beta-globin (hBG) introns were inserted into the human factor IX (hFIX) cDNA in cytomegalovirus (CMV)-regulated plasmids. The resulting construct was then used for further expression analysis in vitro. Seven hFIX-expressing plasmids with different combinations of the two hBG introns and the Kozak element were constructed and used for a systematic expression analysis in cultured Chinese hamster ovary (CHO) cells. In parallel, the hBG intronic sequences were analysed for the presence of possible regulatory elements. All the constructed plasmids resulted in transient expression of the hFIX. However, the coagulation activities varied according to the particular constructs used. Based on the hFIX antigenic assay, a wide range of variation was observed during persistent expression. The second hBG intron appears to be more effective than the first one. The expression level was further increased upon the inclusion of the Kozak element. Sequence analysis has detected several transcription factor binding (TFB) motifs in both of the introns, but with a higher frequency in the second one. Potentials of hBG introns as enhancer-like elements for the expression of the hFIX in cultured CHO cells and a higher activity with respect to the second hBG intron compared to the first one were demonstrated. The larger number of TFBs in the second hBG intron reflects its stronger effect. The results obtained suggest possible synergistic functions of the hBG introns and Kozak on the expression level of hFIX in vitro.

The mycotoxin ochratoxin A inhibits DNA topoisomerase II and induces polyploidy in cultured CHO cells

Ochratoxin A (OTA), a known nephrotoxin and carcinogenic mycotoxin, was investigated to examine its effectiveness to induce cytotoxicity and DNA damage (Comet assay), as well as its possible inhibition of topoisomerase II (topo II) catalytic activity in cultured Chinese hamster ovary (CHO) cells. The analysis of OTA-induced DNA strand breaks as well as the flow cytometric assessment of polyploidy has provided evidence that is consistent with the idea of a mixed mode of action of the mycotoxin: in addition to its genotoxic activity, OTA may also interfere with chromosome distribution during cell division.

Selective and high affinity labeling of neuronal and recombinant nociceptin receptors with the hexapeptide radioprobe [ 3H]Ac-RYYRIK-ol

The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [ 3H]Ac-RYYRIK-ol with exceptionally high specific radioactivity of 94 Ci/mmol. The radioprobe is chemically stable even at 24 °C in ethanol solution for at least 4 days. No significant decomposition of the [ 3H]ligand occurred under the condition of the binding experiments indicating a fine enzymatic stability of the peptide. Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain membrane fractions and recombinant human nociceptin receptor ( hNOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing hNOPr. Specific binding of the compound was reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand binding saturation and homologous displacement experiments indicated the presence of single binding sites in both preparations. Average densities of the [ 3H]Ac-RYYRIK-ol recognition sites were 237 and 749 fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values ( K ds) were determined by three independent way providing identical results. In rat brain membranes K ds of 0.3–1.3 nM were found depending upon the assay type. In homologous competition studies performed on hNOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either with [ 3H]Ac-RYYRIK-ol ( K i 2.8 nM) or with [ 3H](Leu 14)nociceptin (2.3 nM). A number of NOPr and opioid ligands were screened in heterologous displacement experiments and displayed a rank order of affinity profile being consistent with fairly good NOPr selectivity of the sites labeled by [ 3H]Ac-RYYRIK-ol. Taken together, the high molar activity, improved chemical and biological stability and the capability of the selective and high affinity labeling make this novel radioprobe available for further exploring the biochemical pharmacology and receptor-ligand interaction of the NOP receptor.

Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry

A method has been developed for the quantitative profiling of over twenty nucleotides and related phosphorylated species using ion-pair reversed-phase liquid chromatography hyphenated to negative ion tandem electrospray mass spectrometry. The influence of mobile phase pH and ion-pairing agent concentration were assessed to optimise separation and peak shapes. Full quantitative analysis was obtained for the nucleotides by reference to structurally related calibration standards. The developed method was applied to profile changes in nucleotides and related compounds in monolayer cultured Chinese hamster ovary (CHO) cells expressing the β 2 adrenoceptor when exposed to pharmacological stimuli. These experiments demonstrate the potential of the LC–MS/MS method to detect changes in nucleotide drug targets as well as the simultaneous monitoring of levels of other nucleotides.

Cycloheximide and disulfoton are positive in the photoclastogencity assay but do not absorb UV irradiation: another example of pseudophotoclastogenicity?

There is considerable concern regarding the biological plausibility of the response of certain chemicals in the in vitro photoclastogenicity assay, suggesting that this assay is oversensitive and lacks specificity. To explore this further, four coded compounds (aminotriazole, propantheline bromide, cycloheximide and disulfoton) were evaluated for their potential response in a photoclastogenicity assay in cultured Chinese hamster ovary (CHO) cells. None of the four compounds were shown to absorb ultraviolet radiation (UVR) or visible light in the 290- to 700-nm region of the electromagnetic spectrum. A fifth coded compound, tetracycline, which absorbs UVR, was also tested as this has previously been shown to be phototoxic in vitro (3T3-NRU assay) and is cytotoxic, but not genotoxic, at high concentrations in standard 'dark' genotoxicity assays in mammalian cells. The results showed that cycloheximide, disulfoton and tetracycline were clastogenic in CHO cells following UVR exposure (solar-simulated light at 700 mJ/cm(2)) but not in the absence of UVR. Aminotriazole and propantheline were negative in the presence and absence of UVR exposure. Follow-up testing showed that neither cycloheximide nor disulfoton was positive in the 3T3-NRU assay, the standard in vitro regulatory test for phototoxicity, a result consistent with their inability to absorb UVR. These data suggest that both cycloheximide and disulfoton are pseudophotoclastogens, like zinc oxide. Together, these data question the specificity of the in vitro photoclastogencity assay in CHO cells and raises further concern regarding its use for the assessment of chemical photosafety for regulatory purposes. At the very least, a review of the current guidance documents for the photosafety evaluation of pharmaceuticals and cosmetics should be undertaken urgently.

Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC

This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label at the N-terminal (TOAC1-AngII and TOAC0-BK) and internal (TOAC3-AngII and TOAC3-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT1 and B2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. In contrast to internally labeled analogues (TOAC3-AngII or TOAC3-BK), the TOAC1-AngII and TOAC0-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT1 or B2 receptors, respectively. In addition, TOAC1-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC0-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC3-AngII or TOAC3-BK did not provoke any alteration in blood pressure levels. In summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important tool to evaluate peptide–receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT1 and B2 receptors.

A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia

In the serum and cerebrospinal fluid of a patient with recurrent acute episodes of respiratory crises, autonomic symptoms and total insomnia (agrypnia), we identified a novel anti-neural complement fixing antibody directed against GABA B receptor (GABA BR). Patient purified IgG recognized a band of approximately 110 kDa on protein extracts of mouse cerebellum, cortex and brainstem and immunolabelled cultured Chinese hamster ovary (CHO) cells, transfected with human GABA BR1 and rat GABA BR2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both patient purified IgG and anti-GABA BR1 antibody. In order to verify the pathogenic role of these purified antibodies, we injected patient IgG intrathecally into cisterna magna of C57BL/6 mice pre-implanted with EEG electrodes and we observed severe ataxia followed by breathing depression and total suppression of slow wave sleep, as evidenced by EEG recording, in a dose-dependent manner. Immunohistochemistry on brain sections of mice injected with patient IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on Purkinje cells and cerebellar granular layer. After incubation with anti-GABA BR antibody, a marked reduction of receptor immunostaining was found with relative sparing of neuronal architecture. In conclusion we recognized an anti-neuronal autoantibody directed against GABA BR that is associated with autoimmune agrypnia and we showed that our patient purified IgG was able to induce in mice experimental autoimmune agrypnia characterized by a complex neurological syndrome affecting several CNS functions.

小型収束超音波トランスデューサを用いた内視鏡的治療デバイス

In this research, an ultrasonic probe (5.5 mm in diameter), which has a concave PZT transducer at its tip, was fabricated for ultrasonic treatments such as sonoporation and sonodynamic therapy in the human body using a catheter and/or endoscope. Ultrasound has the potential to enhance cytotoxicity of drugs such as porphyrins, a process referred to as sonodynamic therapy, and also to deliver macromolecules such as plasmid DNA, a process referred to as sonoporation. The fabricated probe was then experimentally characterized by measuring the acoustic intensity distribution around the focal point, using a PVDF needle-type ultrasonic hydrophone. When the PZT transducer was driven by a 120 Volts peak-to-peak AC signal at 1.83 MHz, the ultrasound output was successfully focused at the focal point, with a peak intensity of 24.9 W/cm2 (0.87 MPa). Using the fabricated probe, cultured Chinese Hamster Ovary (CHO) cells were exposed to ultrasound (1.83 MHz, continuous wave, peak acoustic pressure of 0.5 MPa) for 2 s in the presence of microbubbles MB-3 and Green Fluorescent Protein (GFP) plasmid DNA. As a result of sonication, the expression of GFP was observed in CHO cells.

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Evaluation of the safety of mixed tocopheryl phosphates (MTP)—A formulation of α-tocopheryl phosphate plus α-di-tocopheryl phosphate

The safety of a formulation of mixed tocopheryl phosphates, (MTP) was evaluated in a series of toxicological tests in vivo using rats, mice and rabbits and in vitro using bacterial and mammalian cell cultures. The tests conducted included an oral LD 50 study, three 28-day oral repeat-dose studies, two dermal toxicity tests, an ocular irritation test, mutagenic potential tests, and chromosomal aberrations tests. MTP consists of mono α-tocopheryl phosphate (TP) and di-tocopheryl phosphate (T 2P) and is intended for use as a dietary supplement and for dermal applications in humans and animals. The dermal and oral LD 50 values of MTP were determined to be >1130 mg/kg bw (918 mg tocopherol equivalents/kg bw) in rabbits and rats, respectively. MTP was not a dermal or eye irritant in rabbits and showed no allergenic potential in mice. In the mutagenicity and genotoxicity studies, MTP did not increased the number of revertants in Salmonella typhimurium or Escherichia coli and did not induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells. When administered daily for 28 days by gavage at doses up to 955 mg/kg bw/day (780 mg tocopherol equivalents/kg bw/day), MTP produced no consistent, dose-dependent adverse effects in rats.

Toxicological safety and stability of the components of an irradiated Korean medicinal herb, Paeoniae Radix

As utilization of medicinal herbs in food and bio-industry increases, mass production and the supply of herbs with a high quality are required. As the use of fumigants and preservatives for herbs is being restricted, safe hygienic technologies are demanded. To consider the possibility of the application of irradiation technology for this purpose, the genotoxicological safety and stability of the active components of the γ-irradiated Paeoniae Radix were studied. The herb was irradiated with γ-rays at a practical dosage of 10 kGy, and then it was extracted with hot water. The genotoxicity of the extract of the irradiated herb was examined in two short-term in vitro tests: (1) Ames test in Salmonella typhimurium; (2) Micronucleus test in cultured Chinese hamster ovary (CHO) cells. The extract of the irradiated herb did not show mutagenicity in the Ames test of the Salmonella reverse mutation assay, and did not show cytogenetic toxicity in the culture of the CHO cells. HPLC chromatogram of paeoniflorin in the irradiated Paeoniae Radix was similar with that of the non-irradiated sample. The quantity of paeoniflorin did not change significantly with irradiation. These results suggest that γ-irradiated Paeoniae Radix is toxicologically safe and chemically stable.