Effect of feeding rate on monoclonal antibody production in a modified perfusion-fed fermentor.

Previously we described a perfusion system for production of high yields of monoclonal antibodies in a fermentor. This system incorporated a cylindrically shaped, stainless steel filter mounted around the stirring shaft for retention of cells within a 1 liter fermentor. Modification of this filter by increasing the pore size from 5 micron to 10 micron decreased its tendency to clog and allowed continuous operation for about 3 weeks. Fresh culture medium, containing 6.5 mg glucose/ml and 3% horse serum, was supplied continually at two different perfusion rates, 850 and 1100 ml/day. Spent culture medium containing monoclonal antibody was harvested concomitantly. Highest cell density (5 X 10(7)/ml) and best antibody yield (1.7 g/l culture per day) were obtained at the higher feeding rate.

Factors affecting cell growth and monoclonal antibody production in stirred reactors.

Environmental and cultural factors that could affect growth and cell viability of mouse-mouse hybridoma cells in culture were investigated. The aim was to determine conditions that could prolong viability and result in increased yields of monoclonal antibodies in stirred reactors. Factors studied included temperature, level of dissolved oxygen, nutrient depletion, and waste product accumulation. Growing cells at temperatures 3-9 degrees lower than optimum (37 degrees C) increased viability but monoclonal antibody production was lowered. A low level of dissolved oxygen (25% air saturation compared to 60% for optimum growth) prolonged cell viability and increased the monoclonal antibody yield by about 50%. Feeding cultures daily to maintain the glucose level above 1 mg/ml and at the same time feeding cells glutamine (150 micrograms/10(6) cells per day) maintained the level of viable cells at 1.7 X 10(6)/ml for at least 9 days and resulted in an antibody yield of 290 micrograms/ml, about a 70% increase over cultures fed either glucose or glutamine alone. Ammonium ion, added to cell populations at levels produced in cultures, stopped cell growth and decreased antibody production. Another waste product, lactic acid, had no toxic effect when added to media at levels found in cultures. These results agree with our suggestion that monoclonal antibody production is enhanced by maintaining cell viability over a prolonged period and provide a base for investigating modes of hybridoma cell propagation in fermentors.

Kinetics of monoclonal antibody production in low serum growth medium.

Factors affecting growth and monoclonal antibody production in vitro by a mouse-mouse hybridoma cell line have been investigated in a series of studies. The goal was to maximize antibody yields and demonstrate that antibodies can be produced efficiently on a large-scale in fermentors. This initial report describes (i) development of a radial immunodiffusion assay for accurate determination of antibody levels in culture, (ii) a culture medium formulation that allowed for reduction in the amount of fetal bovine serum required for good cell growth, and (iii) the kinetics of cell growth and monoclonal antibody production in low-serum media. The radial immunodiffusion assay, employing rabbit anti-mouse IgG antibodies in the immobile phase and the monoclonal antibody (an IgG2a to Rhizobium japonicum cells) as the antigen in the mobile phase, was more reproducible and reliable for determining antibody levels in culture broth than was an indirect enzyme-linked immunosorbent assay. Addition of 0.25% Primatone RL and 0.01% Pluronic F-68 to Dulbecco's modified Eagle medium allowed cells to adapt to growth in medium containing as little as 1% fetal bovine serum; without these additives, 5% serum was the lowest level attained. For the kinetic studies, cells were grown in the low-protein medium in 3 liter spinner flasks. Antibody production occurred during the growth phase, however, significant amounts were also produced during later phases when the cells had stopped growing. Final titers were 100-200 micrograms/ml. It was concluded that maintenance of cell viability is more important than growth rate in production of antibody. This conclusion, confirmed in other studies, has developed into the major underlying strategy employed in subsequent investigations to maximize antibody production in stirred reactors.

Selective adherence of neurons and glial cells from dissociated cerebral and spinal cord microcarrier cultures.

In stationary cultures of dissociated brain and spinal cord grown on microcarriers (MCs), the neuronal and ependymal cells attached to the MCs forming floating aggregates in which they grow in a three-dimensional pattern. The glial and meningeal elements on the contrary, tend to dissociate from the aggregates and adhere to the plastic dish where they divide to form a monolayer. This different behavior of CNS components is not observed in rotating cultures in which all CNS cells remain attached to the MCs and develop into mature floating structures. This cell separation in stationary MC-cultures which is documented here by SEM and immunocytochemistry, may be useful for analysis and evaluation of the metabolic biochemical events of each of the cellular components derived from the same culture.

Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

Factors affecting monoclonal antibody production in culture.

Factors that affect production of monoclonal antibodies (McAb) by a murine cell line were investigated. The goal was to estimate the efficiency of large scale production in stirred reactors. It was found that in batch cultures most McAb was produced after the log growth phase; final yield was 100-200 micrograms/ml. Yields of McAb were increased to 290 micrograms/ml by feeding cells glucose and glutamine. Lactic acid, which was produced in culture as a result of glucose metabolism, had no toxic effect on cells, while another waste product, ammonium ion, was probably accumulated at toxic levels during late stages of cell growth. The hybridoma cell line was propagated in four different systems: fed-batch, semi-continuous, two stage and perfusion. These systems were compared to batch cultures for their effect on cell viability and antibody production. Daily addition of fresh medium (fed-batch propagation) increased antibody productivity from 15 (batch culture) to 27 mg/l of culture/day. In the semi-continuous culture productivity was raised to 34 mg/l/day. Further increase in productivity to a level of 62 mg/l/day was achieved by applying a second batch stage to the semi-continuous culture. A perfusion culturing method was the most effective for production of McAb. Average concentrations of 2.2 X 10(7) live cells/ml and 390 micrograms of antibody/ml corresponding to productivity of 660 mg/l/day were achieved. Serum concentration in the medium was reduced to 0.125% resulting in a specific activity of 0.4 mg of McAb per mg of protein in the cell-free culture broth.

Microcarriers as a culturing system of insect cells and insect viruses.

There is an increasing interest in culturing of insect cells, which are host for arthropod-born (Arbo) viruses. The potential applications of Arbo viruses are in the following two main fields: 1. Medical applications (e.g. preparation of viral vaccines and viral antigens for diagnostic purposes). 2. As bioinsecticides in pest control in horticulture, agriculture and forestry. One of the potential cell substrates for these applications is an anchorage-dependent-mosquito cell line established from embryonic tissues of Aedes aegypti (AA). The following areas were investigated in the reported research with the AA cell line: The AA cells were successfully propagated in microcarrier (MC)-culturing-systems. Of the tested MC's the cellulose-based microgranular MC (DE-53 of Whatman, having an exchange capacity of 2 meq/g) was found to be the best MC. Cells grew in MCs-cells aggregates in submerged spinner culture. The AA-MC's culture was successfully scaled-up to 8 litre culture volume. "Trypsinization" of the AA cells from the MC surface is successfully done by RDB, a dispersion agent from a plant origin (produced and marketed at the author's Institute). Other known dispersion agents (trypsin, collagenase, pronase) failed to disperse the AA cells from the MC. A serum-free medium was developed for culturing the AA cells on the DE-53 MC's. Bovine serum albumin was the main serum substituant in the developed medium. Arboviruses, from the Toga group, were grown in the AA-MC culture. Sindbis virus (from alpha-group) and West Nile virus (from the Flavi group), chronically infected the AA cells, which continuously produce and liberate these two viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammalian cell propagation on derivatized polyacrylamide microcarriers.

A working system for studying the effects of factors involved in the chemical nature of microcarrier on cell attachment, spreading and growth, was established. The system is based on polyacrylamide beads, prepared by the emulsion polymerization technique. Sieved beads of desirable mean diameter were derivatized to generate controlled amounts of primary and tertiary amino groups. These microcarriers were used for propagation of vive different cell strains: BHK, MDCK, CEF, MRC-5 and FS. It was found that BHK cells attach and spread significantly faster on primary amino derivatized beads than those with tertiary amino groups, and at a lower degree of charging. As a result of introducing hydrophobicity to the side chain carrying the primary amino group, higher kinetic rates of BHK cell attachment were obtained. Optimal cell growth of all the tested cells was recorded for the butyl and the hexyl side chains. On the other hand the introduction of hydrophobicity into the polymeric backbone of the microcarrier led to lower cell yields. Primary amino derivatized MCs with an optimal degree of hydrophobicity city exhibit a higher cell yield of MDCK cells (with pronounced epithelial morphology), as compared to the tertiary amino derivatized MCs. CEF and FS cells (with pronounced fibroblast morphology) attained cell yields comparable to those obtained with the tertiary amino derivatized MCs. The diaminohexane derivatized polyacrylamide microcarriers seem to be a potential alternative for the commonly used tertiary amino derivatized micro-carriers.

Role of substrata in determining the growth topology of transformed and nontransformed cells in culture.

Cylindrical DEAE cellulose anion exchangers (DE-53), generally used for chromatography, were found suitable as a substratum for cultivating cells. Embryonic avian and mammalian cells cultured on DE-53 microcarriers (MC) grow in multilayers, while the same embryonic cells when transformed by avian sarcoma virus (ASV) grow in monolayers. These patterns of cell growth differ from those of normal and transformed cells grown on conventional glass or plastic Petri dishes, or on beaded MC. Cells derived from established cell lines such as BHK, HeLa, L-929, MDCK, and VERO grow in monolayer on these MC. A human adenocarcinoma cell line is the only exception growing in a multilayer form. These results indicate that the ability of cultured cells to grow in multilayers, is determined not only by their state of transformation but also by the properties of the support on which they are cultured.

Factors effecting cell attachment, spreading, and growth on derivatized microcarriers: II introduction of hydrophobic elements.

In a previous publication, the authors described the establishment of a working system for studing effects of factors involved in the chemical nature of a microcarrier on cell attachment, spreading, and growth. The first part of the rsearch dealt with the influence of the type and amount of the positively charged groups. In the present article, the authors will describe the effect of the introduction of hydrophobic elements onto primary amino derivatized polyacrylamide microcariers. It was found that cell attachement kinetics were gradually enhanced in parallel to a gradual increase in hydrophobicity via elongation of the hydrocarbon side-chain carrying the primary amino charged group. A threshold effect of the amount of charge required for cell attachment spreading and growth was exhibited on all the tested primary amino derivatized microcarriers. Optimum cell growth was recorded for the butylamine and hexylamine polyacrylamide microcarris. Lowre cell yields were recorded for ethylamine and octylamine derivatives. The location of the introduced hydrophobic element has a profound effect on cell propagation. Introduction of hydrophobicity onto the polymeric backbone of the microcarrier (via copolymerization of hydrophobic comonomer) lead to negative influence on cell attachement and growth yields. Out of the series of derivatized polyacrylamide microcarriers tested, it seems that the hexylamine derivative may be a potential alternative for the commonly used tertiary amine microcariers.

Factors affecting cell attachment, spreading, and growth on derivatized microcarriers. I. Establishment of working system and effect of the type of the amino-charged groups.

A working system for studying the effects of factors involved in the chemical nature of microcarriers on cell attachment, spreading, and growth was established. The system is based on polyacrylamide beads, prepared by the emulsion polymerization technique. Sieved beads of desirable mean diameter were derivatized to generate controlled amounts of primary and tertiary amino groups. These microcarriers were used for the propagation of four different cell strains: BHK, MDCK, CEF, and MRC-5. It was found that BHK cells attach and spread significantly faster on primary amino-derivatized beads than those with tertiary amino groups, and at a lower degree of charging. Cell yields of MDCK cells (with pronounced epithelial morphology) propagated on primary amino-derivatized beads were higher than that obtained for the tertiary amino-derivatized microcarriers. On the other hand, CEF and MRC-5 cells (with pronounced fibroblast morphology) achieved higher cell yields on the tertiary amino-derivatized microcarriers.

A new cellulose-based microcarrier culturing system.

A search for new substrates to be used as microcarriers for culturing mammalian cells was carried out. Commercially available microgranular anion exchange DEAE-cellulose (DE-52 of Whatman) were investigated as microcarriers for anchorage-dependent-cells. Cells from CCL-1 mouse cell line were grown on the investigated microcarriers. Mouse interferon was successfully produced after induction with Sendai virus. Interferon yield per cell was similar to that obtained in monolayer culture.

Interferon-albumin conjugate with conserved biological activity.

The heterobifunctional reagent N-succinilimidyl 3-(2-pyridylthio)propionate (SPDP) was used for the preparation of a disulphide-linked conjugate between Namalwa lymphoblastoid interferon and serum albumin. The linkage of interferon to albumin did not reduce the ability to protect MDBK cells against infection by vesicular stomatitis virus (VSV). The conjugate did not dissociate into free interferon and albumin under conditions prevailing in the assay for interferon activity. The rate of clearance of the interferon-albumin conjugate from the mouse circulatory system was somewhat slower than that of free interferon.

An immobilized hybridoma culture perfusion system for production of monoclonal antibodies.

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 µg/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.

Ion exchange capacity of DEAE microcarriers determined the growth pattern of cells in culture.

Transformed embryonic avian and mammalian cells, as well as cells from established cell lines grow as a monolayer on DEAE-cellulose based microcarriers (MC): the DE-52 and DE-53 MC. Normal, non-transformed, embryonic avian and mammalian cells do not grow on DE-52 MC (having an exchange capacity of 1 meq/g dry material) but grow well on DE-53 MC (having an exchange capacity of 2 meq/g dry material). On DE-53 MC embryonic non-transformed cells grow in multilayers, while embryonic viral-transformed cells and cells from established cell lines grow in a monolayer form. Possible explanations for the differences in cell growth on various MC and variation in the mode of growth in monolayer vs multilayer are discussed. These differences may serve as a valuable tool for separation and distinction between normal and transformed cells. In addition, the described novel MC culturing system provides a support for tridimensional growth on which cell growth mimics the in vivo growth conditions. Therefore, this system is suitable for the study of cell recognition, cell to cell connection and cell orientation.

Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures.

The effect of temperature and O2 saturation on the production of recombinant proteins beta-galactosidase and human glucocerebrosidase by Spodoptera frugiperda cells (Sf9) infected with recombinant Autographa californica nuclear polyhedrosis virus was investigated. The rates of cell growth, glucose consumption, O2 consumption and product expression were measured at temperatures between 22 degrees C and 35 degrees C. The results indicated that possible O2 limitation may be alleviated without compromising the maximum cell yield by lowering the incubation temperature from 27 degrees C to 25 degrees C. The expression level of the recombinant proteins at 27 degrees C was similar to that obtained at 22 degrees C and 25 degrees C; lower protein yields were obtained at 30 degrees C. An increase in temperature from 22 degrees C to 27 degrees C led to earlier production of the proteins and to an increase in the proportion of the product released outside the cells.

Production of recombinant proteins in high-density insect cell cultures.

The effect of the growth phase of Spodoptera frugiperda (Sf9) cells on the production of recombinant proteins (beta-galactosidase and glucocerebrosidase) was investigated. Cells infected with the recombinant Autographa californica nuclear polyhedrosis virus at the late exponential and stationary phases yielded low quantities of expressed protein. Highest enzyme yields were obtained using Sf9 cells from the early exponential phase (0.9 mg beta-galactosidase/10(6) cells and 1.7 microg glucocerebrosidase/10(6) cells). Infection of resuspension of cells collected from various phases of growth in fresh medium resulted in 75% restoration of maximal expression levels. This finding suggested either nutrient limitation or waste product accumulation as the cause of the decrease in productivity at the latter phases of growth. Further experiments revealed that the highest productivity levels could be obtained with cultures of Sf9 cells grown in a fermentor to a cell concentration of 4 x 10(6) mL(-1). The medium needed to be replaced prior to infection with the recombinant virus and supplemented with a mixture of glucose, L-glutamine, and yeastolate ultrafiltrate.