The screening and identification of endogenous retrovirus free CEMPs

The provirus DNA sequence of porcine endogenous retrovirus (PERV) distributed in the pig genome is the major obstacle that restricts the swine as the organ donors in xenotransplantation, and the copy number of PERV varies greatly among different breeds and individuals. In the experiment, 67 healthy, female Chinese Experimental Mini-Pigs (CEMPs) aged at 3–6 months were selected from the Animal Husbandry Station of China Agricultural University, the copy number of PERV and types of envelope protein gene (env) were then investigated by means of PCR analysis and Southern blotting. It is showed that the distribution of types of envelope protein gene in Landrace and CEMPs makes little difference, but the proportion of individuals carrying two types of envelope protein gene (env-A and env-B, which is denoted as env-AB) is much larger than those which carry only one type of envelope protein gene (env-A or env-B). Meanwhile, two endogenous retrovirus free pigs were found for the first time during our research, and the copy number of others is relatively low, which is about 10 to 20. All the results illuminate the genetic diversity of indigenous pig breeds in China and the potential of CEMPs to serve as organ donors in xenotransplantation.     

The screening and identification of endogenous retrovirus free CEMPs

The endogenous retrovirus (ERV) is one kind ofretroviruses that integrated in the genome in the formof provirus and replicates with the proliferation of hostcells. The ERV may play a significant role in the evo-lution, pathology and physiology of animals[1]. Now,proviral sequences of ERV have been found in the ge-nome of many vertebrates, and the release of virionshas also been detected both in vivo and in vitro. Porcine endogenous retrovirus (PERV) embeddedin the genome of pigs belo…     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

Development of a novel,high-throughput screening tool for efficient perfusion-based cell culture process development

Perfusion technology has been successfully used for the commercial production of biotherapeutics, in particular unstable recombinant proteins, for more than a decade. However, there has been a general lack of high-throughput cell culture tools specifically for perfusion-based cell culture processes. Here, we have developed a high-throughput cell retention operation for use with the ambr® 15 bioreactor system. Experiments were run in both 24 and 48 reactor configurations for comparing perfusion mimic models, media development, and clone screening. Employing offline centrifugation for cell retention and a variable volume model developed with MATLAB computational software, the established screening model has demonstrated cell culture performance, productivity, and product quality were comparable to bench scale bioreactors. The automated, single use, high-throughput perfusion mimic is a powerful tool that enables us to have rapid and efficient process development of perfusion-based cell culture processes.     

Exploring the effects of immunity and life history on the dynamics of an endogenous retrovirus

Mammalian DNA is littered with the signatures of past retroviral infections. For example, at least 8% of the human genome can be attributed to endogenous retroviruses (ERVs). We take a single-locus approach to develop a simple susceptible–infected–recovered model to investigate the circumstances under which a disease-causing retrovirus can become incorporated into the host genome and spread through the host population if it were to confer an immunological advantage. In the absence of any fitness benefit provided by the long terminal repeat (LTR), we conclude that signatures of ERVs are likely to go to fixation within a population when the probability of evolving cellular/humoral immunity to a related exogenous version of the virus is extremely small. We extend this model to examine whether changing the speed of the host life history influences the likelihood that an exogenous retrovirus will incorporate and spread to fixation. Our results reveal the parameter space under which incorporation of exogenous retroviruses into a host genome may be beneficial to the host. In our final model, we find that the likelihood of an LTR reaching fixation in a host population is not strongly affected by host life history.     

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103–1112, 2016     

细胞培养工艺条件对抗体异质性影响的研究进展

抗体药物具有靶向明确、副作用小等优势,近年来受到国内外药企的广泛关注。然而,动物细胞大规模培养和抗体质量分析已然成为我国的抗体药物产业化的主要限制因素,尤其是细胞培养工艺条件对抗体异质性的影响非常显著,如何有效通过优化工艺条件来满足抗体药物开发要求成为迫在眉睫的问题。文中简要综述了近年来细胞培养工艺条件对抗体异质性影响的技术进展,并对未来国内抗体药物开发作了展望。     

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Perfusion medium was successfully developed based on our fed‐batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed‐batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ～360 mOsm/kg was desirable resulting in a residual culture osmolality of ～300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell‐specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell‐specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell‐day yielding 1.2 g/L‐day at steady state. This perfusion process was then successfully scaled up to a 100 L single‐use bioreactor with an ATF6 demonstrating similar performance as a 2 L bioreactor with an ATF2. Large volume handling challenges in our fed‐batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug‐and‐play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891–901, 2017     

Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W

Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal-fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41aa) and CHR (34aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-Helix protein exists as a trimer and is highly alpha-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis.     

中国两头乌猪品种内源性逆转录病毒基因研究

目的对5个中国两头乌猪品种(通城猪、东山猪、沙子岭猪、赣西两头乌猪和金华猪)及3个国外品种(大白猪、长白猪和杜洛克猪)猪内源性逆转录病毒(PERV)的核心蛋白(gag)基因、多聚酶(pol)基因、囊膜(env)基因的3个亚型A、B、C,分别从DNA和RNA水平上进行研究,以发现中国两头乌猪品种在异种器官移植中的资源优势。方法利用PCR方法在DNA水平上对PERV基因的三个亚型进行鉴定,并通过半定量PCR方法在RNA水平上检测通城猪和大白猪PERV各亚型在心、肝、脾、肺、肾、肌肉、脂肪、淋巴和脑组织中的表达谱。结果4个华中两头乌猪种中env-AB型为主要PERV亚型,分别占被测总数的92%～100%。在这4个品种中均没有检测到C亚型,金华猪以及3个国外猪种中均检测到了C亚型,病毒亚型种类也更丰富。半定量PCR实验结果显示gag、pol基因在两个品种9个组织中广泛表达,env-A在通城猪的心、肝、肺、脂肪和淋巴组织中表达量较低,env-B在通城猪的心脏和淋巴组织中表达量较低,而env-B在大白猪的肾脏中表达很低,其他所测8个组织中表达量都较高。结论通城猪、东山猪、赣西两头乌猪和沙子岭猪可以做为较佳的异种移植候选供体,具有良好的应用前景。     

重组GM-CSF/IL-3融合蛋白表达菌发酵研究

基因工程菌的发酵技术是基因工程药物大规模生产所必备的关键技术,本文对于重组ＧＭ－ＣＤＦ／ＩＬ－３融合蛋白表达菌株Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）（ｐＦｕ）的生长及产物表达规律进行了探索,在此基础上进行高密度发酵研究,真体最终发酵密度达ＯＤ６００值６０以上,目的产物占菌体总蛋白２５％以上。     

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.     

Hydroxyapatite for use as an animal cell culture substratum obtained by an alternate soaking process

Hydroxyapatite (HAp), the major inorganic component of hard tissues in vivo, was formed in/on a PVA gel. The HAp formation ratio depended on the reaction cycle number, but was independent of the alternate soaking period per cycle. The Ca/P molar ratio of HAp formed at 10 reaction cycles was very close to the theoretical value of HAp, 1.67. CHO-K1 cell adhesion, proliferation and maximum cell density on HAp plates were better on plates formed at two or five reaction cycles using 200 mM CaCl₂ and 120 mM Na₂HPO₄ solutions than on plates formed under other conditions. Furthermore, the adhesion ratio of CHO-K1 cells on HAp plates formed at 10 reaction cycles was about 60% of those at two or five reaction cycles.     

Defining process design space for monoclonal antibody cell culture

The concept of design space has been taking root as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non‐key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product‐related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. Biotechnol. Bioeng. 2010;106: 894–905. © 2010 Wiley Periodicals, Inc.     

Impact of vitrification on granulosa cell survival and gene expression

IntroductionCryopreservation of ovarian tissue is an essential step in Ovarian Tissue Banking. In order to prevent the formation of ice crystals, typically the tissue is slowly frozen using a cryoprotectant. As an alternative the method of ultra-fast freezing by vitrification becomes more attention for freezing ovarian tissue because it has successfully been used for oocytes, embryos and sperm. However the impact of vitrification on granulosa cells, which are an essential part of ovarian tissue is uncertain.AimIn this study, we have therefore analysed the influence of vitrification on the survival rates of granulosa cells, the impact of DMSO or ethylenglycol containing vitrification protocols and investigated to what extent the gene expression of apoptosis- and temperature-sensitive genes changes.Material and methodsWe used the human granulosa cell line KGN as a model for human granulosa cells and determined the survival rate and cell cycle stages by FACS analyses. The change in gene expression was determined by quantitative PCR analyses.ResultsOur results show that vitrification is possible in granulosa cells but it reduces cell viability and leads to fluctuations in the cell cycle. The DMSO containing protocol results in a lower amount of dead cells than the ethylenglycol containing protocol. Gene expression analysis reveals that TNF-alpha expression is strongly increased after vitrification, while other apoptosis or temperature-related genes seem to stay unaffected.ConclusionWe conclude that vitrification influences the viability of human granulosa cells. Furthermore, our results suggest that this could be mediated by a change in TNF-alpha gene expression.     

Changes in wall-bound polysaccharidase activities during the culture cycle of a Rubus fruticosus cell suspension

Several hydrolytic enzyme activities were detected in the wall of developing cells of Rubus truticosus in suspension culture. The corresponding substrates of the enzymes are mostly polysaccharide wall constituents, except for chitinase activity. The activities measured when the enzymes were in the free state or wall-bound showed the positive influence of the cell wall micro-environment. Changes in the activities during a cell culture cycle demonstrated that those enzymes acting on xyloglucans behaved differently from the others, and suggest that xyloglucans undergo modifications in vivo over a longer period of time during the exponential growth phase. The same activities were identified in the culture medium. Endo-1,4-β-d-glucanase activities which depolymerized car☐ymethylcellulose (CMC) and xyloglucans (XG) were assayed viscosimetrically. It was found that XG oligosaccharides exhibited an inhibitory effect on the depolymerization of xyloglucans but not on that of CMC. This suggests that true xyloglucanases are present in the culture of Rubus cells.