Adolfo Castillo

Physicochemical and biological characterization of 1E10 Anti-Idiotype vaccine

Background1E10 monoclonal antibody is 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. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.ResultsHere, we present a comprehensive molecular and immunological characterization of 1E10 produced by the two different production processes in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced 1E10 and bioreactor-obtained 1E10. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models.ConclusionsChanges in 1E10 primary structure like glycosylation; asparagine deamidation and oxidation affected 1E10 structural stability but did not affect the immune response elicited in mice and chickens when compared to 1E10 produced in mice.

Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells

The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.

Adaptation and Selection of NSO Myeloma Cell Lines Producing Recombinant Monoclonal Antibodies in Protein-Free Medium

There are a number of problems associated with the presence of serum in culture medium used for production, including high cost, regulatory considerations and the difficulty in removing serum proteins when purifying the product of interest. By these reasons the use of serum-free (SFM) or protein-free (PFM) media for the culture of genetically engineered mammalian cells offers many advantages.

Purification process development for HER1 extracellular domain as a potential therapeutic vaccine

HER1 is a tumor associated antigen emerging as an attractive target for cancer therapy. In the present study we demonstrated for first time that HER1 extracellular domain can be purified by a downstream process at pilot scale based on immunoaffinity chromatography from bioreactor supernatant of HEK 293 transfectomes. Filtered supernatant was applied to CNBr-activated Sepharose CL-4B with monoclonal antibody anti-human EGF immobilized, followed by three additional chromatographic polishing steps. HER1 extracellular domain was obtained with high purity (>95%), low DNA content, and biological activity.

Cancer vaccine characterization: from bench to clinic.

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS We are demonstrated that this approach could potentially be more efficient and effective for supporting vaccine research and development.

Characterizing the Expression Stability in Different Phenotypes of Recombinant NS0 Myeloma Cell Lines

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.

Semi-Continuous Cultures as a Tool for Cell Line Characterization During Process Development

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.