No abstract

Fed-batch culture currently represents the most attractive choice for large scale production for monoclonal antibodies (MAbs), due to its operational simplicity, reliability, and flexibility for implementation in multipurpose facilities. Development of highly productive cell lines, maximization of cell culture longevity, and maintenance of high specific antibody secretion rates through genetic engineering techniques, nutrition supplementation, waste product minimization, and control of environmental conditions are important for the design of high-yield fed-batch processes. Initially simple supplementation protocols have evolved into sophisticated serum-free multi-nutrient feeds that result in MAb titers on the order of 1-2 g/L. Limited research has been published to date on the effect of various culture parameters on potentially important quality issues, such as MAb glycosylation and stability. Although most fed-batch protocols to date have relied on relatively simple control schemes, increasingly sophisticated algorithms must be applied in order to take full advantage of the potentially additive effects of manipulating nutrient and environmental parameters to maximize fed-batch process productivity.

Molecular imprinting of p-nitrophenyl methylphosphonate, a transition state analogue, in poly[4(5)-vinylimidazole] leads to a polymer which hydrolyses p-nitrophenol acetate at an increased rate and which can then be inhibited by addition of the p-nitrophenol methylphosphonate.

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed.

There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serumfree media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animalsourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1$ 3 · 10 6 viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.

No abstract

As the market for biopharmaceuticals especially monoclonal antibodies (MAbs) rapidly grows, their manufacturing methods are coming under increasing regulatory scrutiny, particularly due to concerns about the potential introduction of adventitious agents from animal-sourced components in the media used for their production in mammalian cell culture. Chinese hamster ovary (CHO) cells are by far the most commonly used production vehicles for these recombinant glycoproteins. In developing animal-component free media for CHO and other mammalian cell lines, the iron-transporter function of serum or human/bovine transferrin is usually replaced by certain organic or inorganic chelators capable of delivering iron for cell respiration and metabolism, but few of them are sufficiently effective. Selenium is a well-known essential trace element (TE) for cell growth and development, and its positive role in biological system includes detoxification of free radicals by activating glutathione peroxidase. In cell culture, selenium in the form of selenite can help cells to detoxify the medium thus protect them from oxidative damage. In this presentation, we describe the discovery and application of a novel function of selenite, that is, as a highly effective carrier to deliver iron for cell growth and function. In our in-house-developed animal protein-free (APF) medium for CHO cells, using an iron-selenite compound to replace the well-established tropolone delivery system for iron led to comparable or better cell growth and antibody production. A high cell density of >10 x 10(6) viable cells/mL and excellent antibody titer of approximately 3 g/L were achieved in 14-day fed-batch cultures in shake flasks, followed by successful scale-up to stirred bioreactors. The preparation of the commercially unavailable iron-selenite compound from respective ions, and its effectiveness in cell-culture performance, were dependent on reaction time, substrates, and other conditions.

A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day(-1). The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day(-1). Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.