Adolfo Castillo

The development of safe, effective, and affordable vaccines has become a global effort due to its vast impact on overall world health conditions. A brief overview of vaccine characterization techniques, especially in the area of high-resolution mass spectrometry, is presented. It is highly conceivable that the proper use of advanced technologies such as high-resolution mass spectrometry, along with the appropriate chemical and physical property evaluations, will yield tremendous in-depth scientific understanding for the characterization of vaccines in various stages of vaccine development. This work presents the physicochemical and biological characterization of cancer vaccine Racotumomab/alumina, a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. Racotumomab was obtained from ascites fluid, transferred to fermentation in stirred tank at 10 L and followed to a scale up to 41 L. The mass spectrometry was used for the determination of intact molecule, light and heavy chains masses; amino acids sequence analysis, N- and C-terminal, glycosylation and posttranslational modifications. Also we used the DLS for the size distribution and zeta potential analysis. The biological analyses were performed in mice and chickens. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced and bioreactor-obtained Racotumomab products. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models. We are demonstrated that this approach could potentially be more efficient and effective for supporting vaccine research and development.

NSO transfected myeloma producing an anti-EGF receptor humanized MAb was cultivated in hollow fiber bioreactors up to production scale (Acusyst-P3/X). Its production rate and metabolic uptakes were compared with a hybridoma, that secreted the murine variant of this product also produced in hollow fiber. The control strategy of the fermentation is discussed. The purification scheme, mainly based on affinity and

Semicontinuous cultures are a powerful tool for cell characterization. However, it is hard to be established at small scale due to the far more complex experimental configuration required. In this work, the use of semicontinuous culture in spinner flasks was explored with daily feeds and extractions, considering that metabolic and growth rates are lower in animal cells under continuous culture regimes. Relatively stabilized conditions were established, characterized by small fluctuations in the concentration of the different metabolic species (glucose, lactate, etc.) at the dilution rates evaluated, and the kinetic parameters (μ, qp, qGlu) were calculated as a function of the dilution rate for each phase. Results obtained were equivalent to those expected for a continuous culture, the semicontinuous operation mode being a much simpler variant. Up to now there are only scarce reports on using semicontinuous cultures as a feasible alternative to simulate the kinetic behavior at statio...

Semicontinuous cultures are a powerful tool for cell characterization. However, it is hard to be established at small scale due to the far more complex experimental configuration required. In this work, the use of semicontinuous culture in spinner flasks was explored with daily feeds and extractions, considering that metabolic and growth rates are lower in animal cells under continuous culture regimes. Relatively stabilized conditions were established, characterized by small fluctuations in the concentration of the different metabolic species (glucose, lactate, etc.) at the dilution rates evaluated, and the kinetic parameters (μ, qp, qGlu) were calculated as a function of the dilution rate for each phase. Results obtained were equivalent to those expected for a continuous culture, the semicontinuous operation mode being a much simpler variant. Up to now there are only scarce reports on using semicontinuous cultures as a feasible alternative to simulate the kinetic behavior at statio...

Valorization of knowledge generated by the fast advance of science and its marketing has led to the so-called “knowledge-based economy” and as a consequence high-technology (HiTech) companies have emerged. Those based in Biotechnology have some specifi c features compared to other HiTech sectors. This paper provides a model to structure the economic management of HiTech Biotechnology organizations in Cuba, considering the national economy and the experience accumulated by several institutions from the Scientifi c Biotech Pole in Western Havana. The model is based on some concepts and proposals, and it addresses key elements, such as: insurance of adequate funding levels for R&D activities and technology replacement, fl exible import and export management, the ability to get into very competitive markets and preservation of a highly qualifi ed workforce. It also demonstrates the feasibility to establish this kind of enterprises in the context and regulations already existing in a non...

Semicontinuous cultures as a tool for characterization of murine hybridoma 1E10 cells. Semicontinuous cultures are a powerful tool for cell characterization. However, it is hard to be established at small scale due to the far more complex experimental configuration required. In this work, the use of semicontinuous culture in spinner flasks was explored with daily feeds and extractions, considering that metabolic and growth rates are lower in animal cells under continuous culture regimes. Relatively stabilized conditions were established, characterized by small fluctuations in the concentration of the different metabolic species (glucose, lactate, etc.) at the dilution rates evaluated, and the kinetic parameters (m, qp, qGlu) were calculated as a function of the dilution rate for each phase. Results obtained were equivalent to those expected for a continuous culture, the semicontinuous operation mode being a much simpler variant. Up to now there are only scarce reports on using semic...

The extensive and labor-intensive process of selection and adaptation of a cell line to culture medium is the biggest limitation of recombinant protein production in mammalian cell culture, due to a very low frequency of the desired clones within a heterogeneously-distributed population of cells. In this study, the kinetic parameters of different clones of a recombinant NS0 myeloma cell line, obtained by serial cloning and adaptation to protein-free medium and secreting the hR3 IgG1 immunoglobulin, were compared. The physico-chemical parameters, biological activity and antibody affinity constant of the antibody produced was determined for each clone. Two culture systems were used to evaluate the effect of culture conditions on antibody structure and functional heterogeneity: adhesion cell cultures in hollow fiber bioreactors and suspension cultures in spinner flasks. A distinct impact of adaptation and selection processes was evidenced on the microheterogeneity of the cell populatio...

The recombinant cell line R3/T16 expresses a “reshaped” Mab against epidermal growth factor receptor (EGF-R) in NSO myeloma. In this study the ability of two culture media to mantain good levels of growth and production of this cell line under serum reduced conditions have been compared and the effect of supplementation of aminoacids and vitamins at 1% of fetal calf

The recombinant cell line R3/T16 expresses a “reshaped” Mab against epidermal growth factor receptor (EGF-R) in NSO myeloma. In this study the ability of two culture media to mantain good levels of growth and production of this cell line under serum reduced conditions have been compared and the effect of supplementation of aminoacids and vitamins at 1% of fetal calf

The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and have been reported by several authors. For this reason understanding the mechanisms involved in the generation of stable and high producer cell lines is very important, especially for those processes based on long-term continuous cultures. In this work we have determined the stability pattern of different recombinant NS0 myeloma cell lines after long term culture, and observed the spontaneous generation of clones with different expression patterns. Moreover, we have characterized two types of clones: unstable and stable with respect to extra- and intracellular light (LC) and heavy (HC) chain expression of recombinant monoclonal antibody, i.e. the expression of typical surface markers for myeloma cell lines. Also, we have compared their respective proteomes using two dimensional gel electrophoresis at the start of cell culture and after 40 generations. it was determined that excess light chain was found in both clones. Eight proteins were associated with the stability profile following a comparison algorithm. The unstable clone showed a decreasing pattern of the expression for these proteins, which are mostly related to protein synthesis and folding, membrane transport, cytoskeletal structure as well as energy production.

A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC–ESI–MS) method was developed for the determination of free and total ropivacaine in human plasma. The work-up procedure involved a simple precipitation of plasma proteins with ...

ABSTRACT When developing cell culture processes based on recombinant mammalian cell lines, researchers faces very often problems related with early selection of that clone, which could better fit with the requirements needed for the future scale-up. Among desired characteristics for newly selected clones are high level and stable expression of desired protein and capability to reach high cell density in protein free-medium, in many cases under nutrient or oxygen limitations. In this work an integrated methodology was developed for the final cell line/culture medium selection. For this aim kinetic parameters were determined in batch cultures and expression stability was studied during long term cultures. An integral multiparametric coefficient was calculated for each clone and five of twelve clones were pre-selected based on this value. The final selection was done based on resistance to apoptosis induction when these cell lines reached limiting conditions at the end of exponential growth phase.

ABSTRACT Production of recombinant therapeutic proteins, especially monoclonal antibodies (Mab), in myeloma cell lines represents a significant segment of the pharmaceutical market, and therefore striving for increased productivity of these lines represents a major investment of resources. The elucidation of biologically important markers for the adaptation of NS0 myeloma cell line to protein-free medium and the recombinant protein production are a major emphasis of our research. These markers could potentially be used in a variety of ways to improve culture conditions, including active approaches to agonize/antagonize important pathways within a medium formulation or diagnostic approaches indicative of improved conditions during the culture. In this work, we used two-dimensional electrophoresis/mass spectrometry and the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 myeloma cell line. Several proteins with differential expression profile were characterized and quantified. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation by proteomic analysis. The same results were obtained using flux balance analysis in a murine metabolic network with selected medium conditions.

In the present work we have carried out a comparative proteomic study of protein expression patterns by two-dimensional gel electrophoresis (2DE) in the pH range of 3–10 between the host cell line NS0 and cell lines that produce different recombinant monoclonal antibodies ...

Although continuous cultures (CC) studies has been recognized as the standard method for cell line characterization, thorough and precise studies makes its use prohibitively costly. Batch cultures have also been used for this purpose due to its simplicity, but because of the constantly changing environment results obtained are reliable only for non limited growth phase.

In this work several clones of a recombinant myeloma NS0 cell that were isolated in protein-free medium and selected by an integrated methodology that takes in account specific production and growth rates, stability of expression and resistance to apoptosis induced ...

Semicontinuous cultures are a powerful tool for cell characterization. However, it is hard to be established at small scale due to the far more complex experimental configuration required. In this work, the use of semicontinuous culture in spinner flasks was explored with daily feeds and extractions, considering that metabolic and growth rates are lower in animal cells under continuous culture regimes. Relatively stabilized conditions were established, char- acterized by small fluctuations in the concentration of the different metabolic species (glucose, lactate, etc.) at the dilution rates evaluated, and the kinetic parameters (μ, qp, qGlu) were calculated as a function of the dilution rate for each phase. Results obtained were equivalent to those expected for a continuous culture, the semicontinuous operation mode being a much simpler variant. Up to now there are only scarce reports on using semicontinuous cultures as a feasible alternative to simulate the kinetic behavior at stat...

Abstract Transforming growth factor alpha (TGFα) is an important epidermal growth factor receptor (EGFR) ligand. Over-expression of both molecules in epithelial tumors has been correlated with poor prognosis and disease progression. Due to the importance of TGFα in ...

Please cite this article in press as: Garcia Duardo K, et al. Assessment of the impact of manufacturing changes on the physicochemical properties and biological activity of Her1-ECD vaccine during product development. Vaccine (2015), a b s t r a c t Vaccine preparations based on the extracellular domain of Her1 (Her1-ECD) have demonstrated, in vitro and in vivo, a potent antimetastatic effect on EGFR + Lewis lung carcinoma model, while associated side effects were absent. The Her1-ECD is a glycoprotein with a molecular weight of 105 kDa and has 11 potential sites for N-glycosylation. Currently Her1-ECD based vaccine has been evaluated in patients with hormone refractory prostate cancer. Her1-ECD molecule used for in clinical trials was obtained from culture supernatant of HEK 293 transfectomes used the protein free culture media and is purified by immunoaffinity chromatography. In order to increase the cell growth and productivity, new defined culture media have been developed (alternative culture media) in Her1-ECD vaccine production process. In this work, a comparability study was performed to evaluate the impact of process changes in the characteristics physic-chemical and biologicals of the Her1-ECD protein and the degree of similitude between both variants. Techniques such as: SDS-PAGE, SEC-HPLC, isoelectric point, peptide mapping, mass spectrometric, SCX-HPLC, oligosaccharide map, ELISA and flow cytometric were used with this aim. Results indicated that this process change decreases the degree of sialylation of the protein but does not affect its biological activity (measured as titers of Abs and recognition for A431 cell line).