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1.
CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor 总被引:1,自引:0,他引:1
R. Valdés N. Ibarra M. González T. Alvarez J. García R. Llambias C. A. Pérez O. Quintero R. Fischer 《Cytotechnology》2001,35(2):145-154
The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow
fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which
is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work
we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg
ml-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value
in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and
antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics
of the mAb harvested from the bioreactor during the 43 days of cultivation.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
2.
Rodolfo Valdés Andrés Tamayo Marcos González Sigifredo Padilla Déborah Geada William Ferro Lorely Milá Leonardo Gómez Rosario Alemán Alberto Leyva Cristina García Otto Mendoza Tatiana Alvarez Lamay Dorta Yanet Villega Dobián Cecilia Hasel Aragón Tatiana González Maylín La O Jorge López 《Biotechnology and Bioprocess Engineering》2012,17(1):145-159
Monoclonal antibody (mAb) production methods (ascites, in vitro technologies, transgenic animals, and dicot or monocot transgenic plants; moss, algae) have been improved since they were first developed in 1975. In this study, we illustrate a summary of a study case in which mice, a hollow fiber system, and tobacco transgenic plants were assessed for the production of mAb for vaccine manufacturing and vaccine production. 相似文献
3.
Neysi Ibarra Abel Caballero Ernesto Gonzlez Rodolfo Valds 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,735(2):314
An immunoaffinity chromatographic method was developed using a mAb immunosorbent to purify recombinant hepatitis B surface antigen (r-HBsAg) from yeast. Elution conditions using a mAb-coated ELISA were improved to select the best conditions to purify r-HBsAg. The optimum results in terms of total quantitative recovery were obtained using 20 mM Tris pH 11.6. An increase in the CB.Hep-1 mAb (anti-HBsAg) useful immunosorbents half-life and in its yield per cycle was obtained when alkaline elution conditions were used. Moreover, the basic conditions do not affect either the antigenic characteristics or the purity or the molecular integrity of r-HBsAg. 相似文献
4.
Alberto Leyva Abrisleida Franco Tatiana González Julio C Sánchez Ivette López Déborah Geada Neyda Hernández Margela Monta?és Iliana Delgado Rodolfo Valdés 《Biologicals》2007,35(1):19-25
An enzyme-linked immunosorbent assay to quantify the mAb CB.Hep-1 during downstream purification process was standardized and validated. This assay is characterized by a short time of incubation at high temperature, allowing the detection of this antibody with high specificity and sensitivity. Detection of antigen-antibody reaction was achieved using a horseradish peroxidase conjugated anti-mouse IgG whose enzyme activity was revealed with o-phenylenediamine substrate. The immunoassay is linear in a range between 3.12 and 50 ng/mL, with a recovery of 98.55-107.62%. According to results, it is possible to estimate the mAb CB.Hep-1 concentration with high precision and reproducibility. The intra- and interassay coefficient of variation ranged from 0.25 to 8.64% and 1.84 to 9.43%, respectively. Significant differences were not observed in the plant-derived antibody quantification by HRP-ELISA and PhoA-ELISA (n=18), demonstrating that plant endogenous peroxidases do not produce interferences in the quantification of this molecule. Therefore, both antibodies can be tested with the same immunoassay with high precision, specificity and accuracy during their respective purification processes without interference of the buffers and sample characteristics. 相似文献
5.
Gómez L Hernández R Ibarra N Valdés R Campos Y Tamayo A Fernández D Figueroa A Alvarez T Montero JA 《Journal of biochemical and biophysical methods》2002,52(3):151-159
Different ligand densities of monoclonal antibody (Mab) CB.Hep-1 were studied during covalent coupling on Sepharose CL-4B for recombinant hepatitis B surface antigen (rHBsAg) immunoaffinity purification. Ligand densities of 2.2, 3.2, 4.2 and 5.2 mg Mab/ml immunosorbents, respectively, were assayed during five cycles of immunoaffinity chromatography (IAC). Adsorption capacities averaged either 3.2 mg/ml (0.57 mg rHBsAg/ml immunosorbent/5.42 mg of total purified protein) or 5.2 mg/ml (0.56 mg rHBsAg/ml immunosorbent/5.05 mg total purified protein). Immunosorbents showed ligand leakage levels below 3 ng Mab/microg rHBsAg. Antigen purity was higher than 95% in all cases. The results suggest that a ligand density (LD) of 3.2 mg Mab/ml immunosorbent should be used for immunoaffinity chromatography because no significant differences were found in the ligand densities studied (P-value=0.012), which saves 40% of CB.Hep-1 immunosorbent manufacturing cost in comparison with 5 mg Mab/ml immunosorbent, which is currently used in large-scale production. 相似文献
6.
Raafat Fahmy Ravikanth Kona Ramesh Dandu Walter Xie Gregg Claycamp Stephen W. Hoag 《AAPS PharmSciTech》2012,13(4):1243-1254
As outlined in the ICH Q8(R2) guidance, identifying the critical quality attributes (CQA) is a crucial part of dosage form development; however, the number of possible formulation and processing factors that could influence the manufacturing of a pharmaceutical dosage form is enormous obviating formal study of all possible parameters and their interactions. Thus, the objective of this study is to examine how quality risk management can be used to prioritize the number of experiments needed to identify the CQA, while still maintaining an acceptable product risk profile. To conduct the study, immediate-release ciprofloxacin tablets manufactured via roller compaction were used as a prototype system. Granules were manufactured using an Alexanderwerk WP120 roller compactor and tablets were compressed on a Stokes B2 tablet press. In the early stages of development, prior knowledge was systematically incorporated into the risk assessment using failure mode and effect analysis (FMEA). The factors identified using FMEA were then followed by a quantitative assessed using a Plackett–Burman screening design. Results show that by using prior experience, literature data, and preformulation data the number of experiments could be reduced to an acceptable level, and the use of FMEA and screening designs such as the Plackett Burman can rationally guide the process of reducing the number experiments to a manageable level.KEY WORDS: failure mode effect analysis (FMEA), Plackett–Burman, quality by design (QbD), quality risk management, roller compaction, tablet and ciprofloxacin 相似文献
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Genetically improved transgenic fish possess many beneficial economic traits; however, the commercial aquaculture of transgenic
fish has not been performed till date. One of the major reasons for this is the possible ecological risk associated with the
escape or release of the transgenic fish. Using a growth hormone transgenic fish with rapid growth characteristics as a subject,
this paper analyzes the following: the essence of the potential ecological risks posed by transgenic fish; ecological risk
in the current situation due to transgenic fish via one-factor phenotypic and fitness analysis, and mathematical model deduction.
Then, it expounds new ideas and the latest findings using an artificially simulated ecosystem for the evaluation of the ecological
risks posed by transgenic fish. Further, the study comments on the strategies and principles of controlling these ecological
risks by using a triploid approach. Based on these results, we propose that ecological risk evaluation and prevention strategies
are indispensable important components and should be accompanied with breeding research in order to provide enlightments for
transgenic fish breeding, evaluation of the ecological risks posed by transgenic fish, and development of containment strategies
against the risks.
Supported by the Development Plan of the State Key Fundamental Research of China (Grant Nos. 2007CB109205 and 2007CB109206),
the National Natural Science Foundation of China (Grant No. 30430540), and the ‘863’ High Technology Project (Grant No. 2006AA10Z141) 相似文献
9.
C Bès L Briant-Longuet M Cerruti P De Berardinis G Devauchelle C Devaux C Granier T Chardès P DeBerardinis 《FEBS letters》2001,508(1):67-74
A systematic exploration of the V(H)2/V(kappa)12-13 variable domains of the anti-CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope-derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti-CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3-like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR-H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti-CD4 peptidomimetics of clinical interest. 相似文献
10.
G C Spagnoli C Ausiello C Palma G Bellone G Ippoliti M Letarte F Malavasi 《Cellular immunology》1991,136(1):208-218
The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1 beta but not IL-2 or interferon-gamma were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43+ cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43+ cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation. 相似文献
11.
Protein-based drugs are the fastest growing class of drugs for the treatment of disease in humans and other animals. However, the current method of producing proteins for pharmaceutical application is predicted to fall short because of population growth and demographic trends. This study characterized human dietary risks using quantitative risk assessment techniques for three pharmaceutical proteins produced in field-grown maize. The three proteins were aprotinin, gastric lipase, and Escherichia coli heat-labile enterotoxin B subunit (LT-B). The human dietary risks from the three proteins inadvertently occurring in food were evaluated using three different exposure scenarios so that potential risks could be compared. The three exposure scenarios ranged in conservatism to evaluate the range of risk between the proteins and scenarios. Risk quotients (RQs) were calculated for all three scenarios to integrate exposure and effect (toxicity). The risk assessments revealed that the most conservative scenario produced higher RQs than the other two scenarios. The dietary risks from scenario 1 for aprotinin were three orders of magnitude greater than for scenario 2, and four orders of magnitude greater than for scenario 3. This risk assessment revealed that dietary risks will vary dramatically and depend on factors such as the specific pharmaceutical protein, protein expression, and exposure scenarios. The assessment also reinforced the need for case-by-case assessments. 相似文献
12.
A bactericidal monoclonal antibody elicits a change in its antigen, OspB of Borrelia burgdorferi, that can be detected by limited proteolysis 总被引:1,自引:0,他引:1
Katona LI Ayalew S Coleman JL Benach JL 《Journal of immunology (Baltimore, Md. : 1950)》2000,164(3):1425-1431
mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage. 相似文献
13.
Tijen Talas-Oğraş 《Acta Physiologiae Plantarum》2011,33(3):647-657
Advances in recombinant DNA technology have created advantages for the development of plants with high agro-economical values.
Since the production of transgenic plants, some issues concerning the safe use of these plants and their products have been
under debate throughout the world. In this respect, the potential risks and benefits of transgenic plants need to be evaluated
objectively. Risk assessment of transgenic crops is a basic prerequisite for monitoring the possible risks that could arise
upon the release and use of transgenic plants. To get a meaningful tool for decision making, risk assessment needs to be carried
out in a scientific sound and transparent manner. There are specific governmental regulations in many countries for the safety
assessment of genetically modified (GM) crops. Furthermore, there are some international agreements, which regulate the cultivation
and commercialization of transgenic plants and their derivatives. Internationally accepted risk assessment strategies have
been performed to evaluate the safe use of a large variety of GM crops. The main objectives of these regulations and risk
assessment strategies are focused to protect human/animal health and the environment. 相似文献
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15.
Akiko Ishii-Watabe Akihiko Hirose Noriko Katori Norikata Hashii Susumu Arai Hirotoshi Awatsu Akira Eiza Yoshiaki Hara Hideshi Hattori Tomomi Inoue Tetsuya Isono Masahiro Iwakura Daisuke Kajihara Nobuo Kasahara Hiroyuki Matsuda Sei Murakami Taishiro Nakagawa Takehiro Okumura Takeshi Omasa Shinya Takuma Iyo Terashima Masayoshi Tsukahara Maiko Tsutsui Takahiro Yano Nana Kawasaki 《AAPS PharmSciTech》2015,16(5):993-1001
Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.KEY WORDS: biologics, manufacturing technology, quality risk management, regulatory science, single-use system 相似文献
16.
Ran Zhang Man Rao Chuan Li Jingyuan Cao Qinglin Meng Min Zheng Meili Wang Yunping Dai Mifang Liang Ning Li 《Transgenic research》2009,18(3):445-453
Hepatitis A virus (HAV) is a wide spread pathogenic agent and is the common cause of acute Hepatitis A worldwide. Passive
immunization of HAV plays an extremely important role in post-exposure prophylaxis with clinical applications often requiring
large amounts of antibody. As an alternative to the in vitro production of recombinant proteins, expression of monoclonal
antibodies (mAbs) in the milk of transgenic animals is currently used being associated with low production costs and high
activity. In this paper, eight founder lines of transgenic mice were generated by co-microinjection of the two cassettes encoding
the heavy- and light-chains of a neutralizing anti-HAV antibody, respectively. The expressed heavy- and light-chains of the
mAb were correctly assembled and modified in the mammary gland as detected by western blotting. High expression levels of
the antibody were achieved during the lactation period and found to be independent of the copy numbers of integrated transgenes.
The highest level was up to 32.2 mg/ml. The binding specificity and neutralizing activity of the expressed mAb were assayed
by ELISA and neutralizing test, showing that it is capable to neutralize the JN strain of Hepatitis A virus efficiently. Therefore,
our results suggest that a large-scale and efficient production of the anti-HAV mAb in the milk of transgenic farm animals
would be feasible in the future. 相似文献
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With the emergence of transgenic technologies, new ways to improve the agronomic performance of crops for food, feed, and processing applications have been devised. In addition, ability to express foreign genes using transgenic technologies has opened up options for producing large quantities of commercially important industrial or pharmaceutical products in plants. Despite this high adoption rate and future promises, there is a multitude of concerns about the impact of genetically modified (GM) crops on the environment. Potential contamination of the environment and food chains has prompted detailed consideration of how such crops and the molecules that they produce can be effectively isolated and contained. One of the reasonable steps after creating a transgenic plant is to evaluate its potential benefits and risks to the environment and these should be compared to those generated by traditional agricultural practices. The precautionary approach in risk management of GM plants may make it necessary to monitor significant wild and weed populations that might be affected by transgene escape. Effective risk assessment and monitoring mechanisms are the basic prerequisites of any legal framework to adequately address the risks and watch out for new risks. Several agencies in different countries monitor the release of GM organisms or frame guidelines for the appropriate application of recombinant organisms in agro-industries so as to assure the safe use of recombinant organisms and to achieve sound overall development. We feel that it is important to establish an internationally harmonized framework for the safe handling of recombinant DNA organisms within a few years.This is IMTECH Communication No. 038/2005. 相似文献
20.
Denizhan Yilmaz Hamidreza Mehdizadeh Dunie Navarro Amar Shehzad Michael O'Connor Philip McCormick 《Biotechnology progress》2020,36(3):e2947
Monoclonal antibodies (mAbs) are biopharmaceuticals produced by mammalian cell lines in bioreactors at a variety of scales. Cell engineering, media optimization, process monitoring, and control strategies for in vitro production have become crucial subjects to meet increasing demand for these high value pharmaceuticals. Raman Spectroscopy has gained great attention in the pharmaceutical industry for process monitoring and control to maintain quality assurance. For the first time, this article demonstrated the possibility of subclass independent quantitative mAb prediction by Raman spectroscopy in real time. The developed model estimated the concentrations of different mAb isotypes with average prediction errors of 0.2 (g/L) over the course of cell culture. In situ Raman spectroscopy combined with chemometric methods showed to be a useful predictive tool for monitoring of real time mAb concentrations in a permeate stream without sample removal. Raman spectroscopy can, therefore, be considered as a reliable process analytical technology tool for process monitor, control, and intensification of downstream continuous manufacturing. The presented results provide useful information for pharmaceutical industries to choose the most appropriate spectroscopic technology for their continuous processes. 相似文献