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1.
A coiled tube membrane reactor was developed for the cultivation of mouse-mouse hybridoma cells producing monoclonal antibodies. The cell and antibody concentrations obtained in the membrane reactor were higher than that obtained in a batch spinner flask without a membrane. A mathematical model has been developed to describe the membrane transport coupled growth and product formation, and the physical and kinetic constants of the system were determined. 相似文献
2.
The number of Hybridomes obtained from various sources rapidly increases at present. Clones producing monoclonal antibodies to influenza virus A/USSR/090/77 and to VEE-230 are generated in the laboratory of the Institute of Virology (Academy of Sciences of the USSR). The present work is devoted to the study of Hybridome karyotype by means of C-method for chromosome staining with the aim to reveal specific characteristics of these lines. Results of the investigation have shown that the count of chromosomes together with an examination of their C-staining picture permit proving a hybrid nature of the clones and identifying various Hybridomes by chromosome markers. 相似文献
3.
Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed.Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted.Abbreviations MCAB
monoclonal antibodies
- PBS
phosphate buffered saline
- RT
room temperature
- SDS
sodium dodecyl sulphate 相似文献
4.
Electrical stimulation was applied to hybridoma cells in order to activate metabolic activities and increase the monoclonal antibody production. Hybridoma cells that produce monoclonal antibody to adenosine 3':5'-cyclic monophosphate were placed on a transparent glass electrode immersed in medium and subjected to electric pulses (pulse shape, alternating rectangular; field strength, 4 X 10(3) V X m-1; frequency, 5 kHz; pulse mode, 0.5 min application and 4.5 min pause). After 48 h of incubation, the concentration of lactic acid in the medium reached 8.4 mM, approx. 30% higher than that obtained without electric stimulation. Similarly, cell growth rate was promoted by the electric stimulation, reaching a maximum stimulation after 40 h. When the hybridoma was cultured for 48 h with electrical stimulation, the antibody concentration in the medium reached 22.3 microgram X ml-1, approx. 10% higher than the control, with a concomitant 16% increase in cell concentration. Longer periods of electric pulse application, however, caused an inhibitory effect on the hybridoma growth. The most probable cause of the inhibition are reactive oxygen species such as superoxide and hydrogen peroxide, which are inevitably generated by electrolysis. The presence of superoxide dismutase (EC 1.15.1.1) reduced the inhibitory effects. In conclusion, metabolic activities including monoclonal antibody production were activated by the electrical stimulation. 相似文献
5.
Juan Dubrot Aitziber Portero Gorka Orive Rosa María Hernández Asis Palazón Ana Rouzaut Jose L. Perez-Gracia Sandra Hervás-Stubbs Jose Luis Pedraz Ignacio Melero 《Cancer immunology, immunotherapy : CII》2010,59(11):1621-1631
Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment
the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins
is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets.
In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration
of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules
containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced
by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and
thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of
the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal
administration of the purified antibodies. The rat–mouse hybridoma cells did not engraft as tumors in immunocompetent mice,
while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the
strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction
of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof
of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody
to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb. 相似文献
6.
Sally S. Seaver Ph.D. 《Cytotechnology》1992,9(1-3):131-139
We are interested in determining the range of variants present in a cell population that can actually be isolated. We have
used subcloning and sublining to search for variants with increased antibody stability, increased cell line stability to freezing
and defrosting, increased cell population viability, increased antibody production and the ability to grow in simpler media.
This paper presents the case histories of several different hybridoma cell lines which required some property changed before
they became production ready clones. We found that switching the class of an antibody from IgG3 to IgG1 did increase its stability, decrease its tendency to aggregate and allowed it to be used in a commercial diagnostic kit.
We could isolate subclones that produced twice the level of antibody with a frequency of 1–3%. It was straight forward to
isolated clones that were stable to freezing and defrosting or grew in a simpler media. We were not successful in increasing
the maximum viability of a cell line. In conclusion, we have found that any population of hybridoma cells has natural variants
with significantly enhanced properties that can be isolated. 相似文献
7.
Stacey Spencer Deidra Bethea T. Shantha Raju Jill Giles-Komar Yiqing Feng 《MABS-AUSTIN》2012,4(3):319-325
The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble. 相似文献
8.
《MABS-AUSTIN》2013,5(3):319-325
The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble. 相似文献
9.
Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies
An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new nutrient-split feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l-1 was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system.Abbreviations: cGlc – glucose concentration, mmol l-1; cGln – glutamine concentration, mmol l-1; cAmm – ammonia concentration, mmol l-1; cLac – lactate concentration, mmol l-1; cMAb – MAb concentration, mg l-1; D – dilution rate, d-1; Di – dilution rate in the inner chamber of the membrane dialysis reactor, d-1; D0 – dilution rate in the outer chamber of the membrane dialysis reactor, d-1; q*FB,Glc – volume specific glucose uptake rate related to the fixed bed volume, mmol lFB
-1 h-1; q*FB,Gln – volume specific glutamine uptake rate related to the fixed bed volume, mmol lFB
-1 h-1. 相似文献
10.
van der Pol JJ Machnik M Biselli M Portela-Klein T de Gooijer CD Tramper J Wandrey C 《Cytotechnology》1997,24(1):19-30
The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored
on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method
(ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns.
After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the
column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure
was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and
antibody concentrations measured by HPLC was linear.
A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the
circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre,
situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system
was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter,
which had a surface of 4.8 cm2, resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical
device functioned without failure during this long-term culture.
The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous
increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium
stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth
rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates
also increased in the temperature range between 38 to 40 °C.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
11.
As the interest in weak-affinity antibodies has been widened by their introduction to various analytical techniques such as HPLC, capillary electrophoresis and biosensors, there has been a need for new screening/monitoring methods. In this study, weak-affinity chromatography was adopted to screen/monitor directly for monoclonal antibodies in ascites. Monoclonal antibodies against a carbohydrate antigen (maltohexaose) were used to evaluate this approach. In short, malthohexaose was immobilized on an HPLC support in such a configuration to allow, during HPLC, retardation of weak monoclonal antibodies. Based on the retention, the affinity or the avidity, as determined by the presence of multiple binding of the monoclonal antibody towards antigen, can be estimated. In this way it is possible to select clones of hybridomas that produce desired weak monoclonal antibodies. Adjustments in temperature (10-20 degrees C) were used to moderate the retention and hence affinity of the weak monoclonal antibodies during chromatography. 相似文献
12.
13.
The purpose of this study was to investigate hybridoma growth and monoclonal antibody formation in a flat sheet membrane bioreactor. The effects of several different molecular weight cutoff membranes on growth and antibody formation were investigated. Nutrient and toxic product diffusion through the membranes were quantified, and the kinetic and physical constants of the system were determined. 相似文献
14.
Light and electron microscopic studies of cellular localization of oPL with monoclonal and polyclonal antibodies. 总被引:2,自引:0,他引:2
F B Wooding G Morgan I A Forsyth G Butcher A Hutchings S A Billingsley P D Gluckman 《The journal of histochemistry and cytochemistry》1992,40(7):1001-1009
Accurate knowledge of placental lactogen localization is fundamental to any hypothesis of its synthesis and secretion. We used locally generated monoclonal and polyclonal antibodies from three separate sources to localize ovine placental lactogen immunoreactivity on light and electron microscope Lowicryl K4M sections of ovine placentomes of 97-145 days of gestation, using immunogold techniques. All antibodies demonstrated that immunoreactivity was exclusively localized in the trophoectoderm binucleate cell Golgi body and granules and in granules in the syncytium derived from binucleate cell migration. No evidence was found to support a recent claim that monoclonal antibodies to oPL that were produced in Canada indicated a predominant localization of ovine placental lactogen to uninucleate trophectodermal cells. 相似文献
15.
《Biochemical Engineering Journal》2000,4(2):153-168
Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359–368; E. Suzuki, D.F. Ollis, Biotechnol. Bioeng. 34 (1989) 1398–1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61–69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture. 相似文献
16.
《Journal of biotechnology》1988,7(3):179-184
In biotechnology, the interest in mass cultures of animal cells rises constantly; thus knowledge of the conditions influencing the cultures becomes important. Suspension cell cultures in bioreactors are subject to considerable shear forces, when stirring devices are employed. In order to get reliable data on the influence of shear forces on proliferation and antibody production of hybridoma cells, we tested cells, producing monoclonal antibodies against mitochondrial creatine kinase (Mi-CK), under laminar flow conditions in a rotating viscosimeter. Flow conditions in the annular gap were characterized by measuring the speed of revolution of the inner cylinder and the torque. From these data the shear stress could be determined. To confirm the laminar flow condition the velocity profile was determined by laser Doppler anemometry (LDA). After exposure to shear stress, cells were tested for viability, growth rate, antibody production as well as for the glucose uptake and lactate production rates. The data showed that cell death increases as a function of shear stress. The cells, remaining viable after exposure to shear stress showed growth and production rates similar to untreated cells. 相似文献
17.
A fixed bed perfusion system for hybridoma cell immobilization is presented. The system consists of a culturing vessel (300 ml total volume) in which polyurethane (PU) sponges in the form of small cubes of about 5 mm sides are packed. Cells are immobilized by physical entrapment in the foam matrix. By entrapment of the cells in the pores of the matrix high cell concentration can be maintained in a mechanically protected environment. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber (700 ml total volume).Medium flow rate, feeding rate, dissolved oxygen, pH, nutrient uptake and waste product formation can be easily monitored and controlled. Steady state conditions are established with medium dilution rates of 1.0–1.5 reactor volume per day. The steady state is characterized by a constant cell density, constant culture volume and constant glucose and lactate levels. Cell-free supernatant is collected continuously in a cold room adjacent to the 37°C culture room. Monoclonal antibodies (MAb) are produced at a concentration of 150–200 g/ml for several weeks. An important feature of the system is the capacity to maintain a population of cells after the growth phase in a non-proliferating state for extended time periods expressing high titers of MAb.Abbreviations DO
Dissolved Oxygen
- FBS
Fetal Bovine Serum
- FBR
Fixed Bed Reactor
- MAb
Monoclonal Antibody
- PU
polyurethane 相似文献
18.
Summary Mouse hybridoma cells were succesfully cultivated for more than 640 hours in the interparticle spaces of a tubular reactor packed with spherical glass beads. The maximum monoclonal antibody (MAb) concentration attained was 110 mg/l and a viable cell density in the order of 1 × 107 cells/ml was achieved. A productivity per reactor void volume of 5.2 mg MAb/hr/l was obtained, which is comparable to the best systems currently in use. 相似文献
19.
20.
In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124) to obtain increased production of monoclonal antibodies (IgG2a), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface-associated antibodies and a fluorescence-activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry. 相似文献