Advances and drawbacks of the adaptation to serum-free culture of CHO-K1 cells for monoclonal antibody production

Currently, mammalian cell technology has become the focus of biopharmaceutical production, with strict regulatory scrutiny of the techniques employed. Major concerns about the presence of animal-derived components in the culture media led to the development of serum-free (SF) culture processes. However, cell adaptation to SF conditions is still a major challenge and limiting step of process development. Thus, this study aims to assess the impact of SF adaptation on monoclonal antibody production (mAb), identify the most critical steps of cell adaptation to the SF EX-CELL medium, and create basic process guidelines.. The success of SF adaptation was dependent on critical steps that included: accentuated cell sensitivity to common culture procedures (centrifugation, trypsinization); initial cell concentration; time given at each step of serum-reduction; and, most importantly, medium supplements used to support adaptation. Indeed, only one of the five supplement combinations assessed (rhinsulin, ammonium metavanadate, nickel chloride and stannous chloride) succeeded for the CHO-K1 cell line used. This work also revealed that the chemically-defined EX-CELL medium benefits mAb production in comparison with the general purpose Dulbecco's Modified Eagle's Medium, but the complete removal of serum attenuates these positive effects.The authors acknowledge funding and support from the Portuguese Foundation for Science and Technology (FCT), namely grant ref SFRH/BD/46661/2008 for Maria Elisa Rodrigues and SFRH/BD/46660/2008 for Ana Rita Costa

Evaluation and comparison of two-liquid phase hydrolytic reaction in a stirred tank and membrane biocatalytic reactor

The introduction of a second liquid phase as a means to improving the productivity of bioreactions involving poorly water soluble compounds is now a fairly well established technique. It is particularly applicable for the generation of chiral intermediates via enzyme catalysed hydrolytic resolution. The work presented in this thesis attempts to address issues which will aid in the understanding of elements involved with the commercial development of such processes. Hydrolytic reactions can be facilitated by both esterases and lipases. In this study a purified pig liver esterase and a crude porcine pancreatic lipase were found to catalyse hydrolysis of a model substrate benzyl acetate, in a two liquid phase medium, in a stirred tank reactor. In previous study pig liver esterase was found to catalyse hydrolysis solely in the aqueous phase, in contrast porcine pancreatic lipase was found to catalyse reaction at the interface. In the stirred tank reactor, the activity of the esterase was dependent on the mass transfer conditions in the reactor as influenced by operating variables of phase ratio and agitation rate. Using data from previous studies and that obtained in this study mass transfer conditions were evaluated and optimum conditions of operation in the stirred tank reactor were identified. The optimal mass transfer conditions in the reactor did not support large aqueous phase concentrations of the pig liver esterase. The lipase activity was influenced by the amount of interface in the reactor. The optimal interfacial operating conditions supported large aqueous phase concentrations fo the lipase preparation at maximum activity. The esterase was prone to interfacial effects and product inhibition and thus had limited stability. Resultingly overall conversion of substrate was low. In contrast the lipase was comparatively more stable and the overall conversion of substrate using lipase as the catalyst was much greater. Although reaction could be carried out in the stirred tank reactor, difficulties were forseen in the downstream separation of reactor contents for product recovery and catalyst reuse. Conditions in the stirred tank reactor, necessary to maintain optimal enzyme activity, led to the irreversible destruction of the enzyme. To overcome some of these problems a further reaction device was studied, a membrane bioreactor. Fundamentally the two reactors differed in the way in which contact of the two phases was facilitated, in the stirred tank reactor this was by dispersion, in the membrane reactor by contact across a membrane surface, and the enzyme location, in the stirred tank reactor solubilised in the aqueous phase and in the membrane bioreactor immobilised onto the membrane surface. The lipase catalysed hydrolysis was compared in the membrane reactor with its hydrolysis in the stirred tank reactor. The activity of the enzyme in the membrane reactor was severely limited in comparison to activity in the stirred tank reactor. Limitations were greatest at high enzyme loads. Several ideas were proposed as to why this might be. Although the activity of the enzyme was limited, the stability was much improved. The time required to achieve the same degree of conversion in the membrane reactor as achieved in the stirred tank reactor was greater. The membrane reactor facilitated phase separation throughout the course of the reaction and enzyme was reused in several subsequent reactions with some loss of activity between runs. The results and methods used to obtain them provide useful tools and guidelines and a scientific basis which can be applied to the evaluation of similar reaction systems in order to identify the most effective process option

Evaluation of the OSCAR (TM) system for the production of monoclonal antibodies by CHO-K1 cells

Biopharmaceutical production of complex recombinant protein therapeutics currently relies on mammalian cells. The development of high-yielding stable cell lines requires processes of transfection, selection and adaptation. With several technologies available, selection has been most frequently based on dihydrofolate reductase or glutamine synthetase systems, which can be very time-consuming. Due to the pressure to reduce development costs and speed up time to market, new technologies are emerging, as the promising OSCAR™ expression system that could provide more rapid development of high-yielding stable cell lines than the traditional systems. However, further evaluation of its application in a wider range of cell types and media is still necessary. In this study, application of OSCAR™ for the transfection of a CHO-K1 cell line with a monoclonal antibody was evaluated. OSCAR™ was reasonably fast and simple, without negative impact on cell growth characteristics. However, minigene selection was critical, with only pDWM128 working for the cell line assessed. Initial relatively high levels of production decreased significantly in the first few weeks of passing, remaining relatively stable although with low yield thereafter. The results suggest that more work is required to develop methodologies and prove that OSCAR™ has significant value to the bioproduction industryFundação para a Ciência e a Tecnologia (FCT

Superior Suppression of ErbB2-positive Tumor Cells by a Novel Human Triparatopic Tribody

Downregulation of the epidermal growth factor receptor family of receptors is improved by combining different antibodies to noncompetitive epitopes. For ErbB2/HER2 this has already been translated into clinical practice by using a combination of trastuzumab and pertuzumab. Moreover, cocktails of 2 or 3 anti-epidermal growth factor receptor antibodies show an enhanced downregulation of the receptor due to the induction of matrix formation. A more efficient method for inducing matrix formation and receptor downregulation might include the use of trispecific reagents. A triparatopic Tribody consisting of 3 noncompeting ErbB2 binders was compared with equivalent trivalent monoparatopic counterparts, as well as to a cocktail of 3 monoclonal antibodies for its effects on downregulation of the ErbB2 receptor's kinase activity and survival of several ErbB2-expressing cancer cell lines. The triparatopic Tribody was significantly more efficient in downregulating ErbB2 and inhibiting tumor cell growth than either the control monoparatope tribodies or the combinatorial treatment with the 3 different parental antibodies on all the tested tumor cells, including trastuzumab-resistant cell lines. The enhancement of effectivity was dependent on all 3 binding moieties. Because the novel Tribody allows reduction of the costs of production (as only 1 construct provides the antitumor effects of 3 antibodies) and has an intermediate molecular size (∼100 kDa) well suited for both tumor penetration and acceptable half-life, it has the potential to become a precious tool for therapeutic use particularly in trastuzumab-resistant cancer patients

Evaluation of macroporous and microporous carriers for CHO-K1 cell growth and monoclonal antibody production

The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for small-scale monoclonal antibody (mAb) production by CHO-K1 cells. Cultures (5 mL) of microporous Cytodex 3 and macroporous CultiSpher-S carriers were performed in vented conical tubes and subsequently scaled-up (20 mL) to shake-flasks, testing combinations of different culture conditions (cell concentration, microcarrier concentration, rocking methodology, rocking speed and initial culture volume). Culture performance was evaluated considering mAb production and cell growth at the phases of initial adhesion and proliferation. The best culture performances were obtained with Cytodex 3, regarding cell proliferation (average 1.85 ± 0.11 x 106 cells/mL against 0.60 ± 0.08 x 106 cells/mL for CultiSpher-S), mAb production (2.04 ± 0.41 µg/mL against 0.99 ± 0.35 µg/mL for CultiSpher-S) and culture longevity (30 days against 10-15 days for CultiSpher-S), probably due to the collagen-coated dextran matrix that potentiates adhesion and prevents detachment. The culture conditions of greater influence were rocking mechanism (Cytodex 3, pulse followed by continuous) and initial cell concentration (CultiSpher-S, 4x105 cells/mL). Microcarriers proved to be a viable and favorable alternative to standard adherent and suspended cultures for mAb production by CHO-K1 cells, with simple operation, easy scale-up and significantly higher levels of mAb production. However, variations of microcarrier culture performance in different vessels reiterate the need for optimization at each step of the scale-up process.The authors acknowledge funding and support from the Portuguese Foundation for Science and Technology (FCT), namely grant ref SFRH/BD/46661/2008 for Maria Elisa Rodrigues and SFRH/BD/46660/2008 for Ana Rita Costa