Continuous purification of monoclonal antibody using periodic counter-current chromatography

Integrated and continuous processing of antibody drugs offers several advantages over traditional batch processing in the biotechnology industry. The flexibility of periodic counter-current (PCC) design is performed in the selection of residence time and column numbers on the capture process. In this study, we investigated the association of residence time and product recovery in the downstream PCC purification. A practical operation of PCC as a continuous capture purification step has been applied to 5L perfusion culture, 5L concentrated fed-batch culture, and 50L fed-bath culture. Using an empirical model for the protein breakthrough curve, residence time (RT) was evaluated and the loading flow rate was adjusted to achieve a target RT of 2.25 minutes for monoclonal antibody (mAb). The sample load volume for each column switching was set on 50 and 58% breakthrough curves, mAb recovery was 88 .4% and 88.9%, and buffer consumption was decreased to under half that of the batch process. Overall, more than 40 grams of purified antibody is obtained in 24 hours using a PCC purification system. Comparison of qualities of mAb analyzed by UPLC and reverse phase chromatography show that glycan profiles and purity are quite similar between antibodies obtained from PCC and batch purification, whereas the acidic variants of mAb purified by PCC is higher than that purified by batch mode. The advantages of a continuous downstream capture step are highlighted for our case study in comparison with the existing batch chromatography processes

Periodic counter-current chromatography for continuous purification of monoclonal antibody

Integrated and continuous processing of antibody drugs offers several advantages over traditional batch processing in the biotechnology industry. The flexibility of periodic counter-current (PCC) design is performed in the selection of residence time and column numbers on the capture process. In this study, we investigate the association of residence time and product recovery in the downstream PCC purification. A practical operation of PCC as a continuous capture purification step has been applied to 50L feed-bath culture, 5L perfusion culture and 5L concentrated feed-batch culture. Protein breakthrough curve was determined for the appropriate column switching strategy. Using an empirical model for the protein breakthrough curve, residence time (RT) was evaluated and the loading flow rate was adjusted to achieve a target RT of 2.25 minutes for monoclonal antibody (mAb). The sample load volume for each column switching was set on 50-58% breakthrough curves, mAb recovery was 83-92%, and buffer consumption was decreased to under half that of the batch process. Overall, 1.0 to 1.5 gram mAb was obtained for per milliliter resin in 24 hours using a PCC purification system. We used size exclusion-high performance liquid chromatography to confirm composition and masses of our fragment samples. Comparison of qualities of mAb analyzed by UPLC and reverse phase chromatography show that glycan profiles and purity are quite similar between PCC and Avant purification, whereas that for acidic variants are different, the acidic variants of mAb purified by PCC is higher than that purified by Avant. The advantages of a continuous downstream capture step are highlighted for our case study in comparison with the existing batch chromatography processes. The use of PCC improves the higher resin capacity utilization and lower buffer consumption

Method of laser casting copper-based composites

US6723278Granted Paten

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

<div><p>Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects <i>in vitro</i>. To evaluate the effects of HDGF on tumour growth and metastasis <i>in vivo,</i> syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.</p> </div

Effects of HDGF overexpression or gene silencing on tumorigenesis of melanoma cells <i>in vitro</i> and <i>in vivo.</i>

<p>(A) A representative western blot demonstrating HDGF protein expression in B16–F10 melanoma cells infected with Ad-GFP, Ad-HDGF shRNA and Ad-HDGF. The β-actin was used as controls. (B) Representative photos and quantification of colony formation ability assay (from 3 experiments). (C) Weights of tumors isolated from C57BL/6 mice. Melanoma cells infected with recombinant adenovirus for 24 hrs were subcutaneously implanted into mice over 28 days. Tumors were then excised and weighted (n = 10/each group). (D) The representative profile of Ki-67 expression in tumor sections from mice inoculated with B16–F10 melanoma cells infected with Ad-GFP, Ad-HDGF and Ad-HDGF shRNA. Bar chart shows proliferation index of Ki-67 positive cells in tumor tissues (n = 6/each group). All data are expressed as mean ± SEM. *<i>P</i><0.05 compared to Ad-GFP-treated groups, one-way ANOVA with post-doc analysis.</p

Schematic representation of HDGF stimulated tumor progression in melanoma.

<p>Schematic representation of HDGF stimulated tumor progression in melanoma.</p

Expression level of HDGF and effects of HDGF on tumorgenicity of human melanoma cells <i>in vitro.</i>

<p>(A) The gene expression of HDGF in human epidermal melanocyte (HEMn) and melanoma cell lines (A375, A2058, MEL-RM and MM200) was measured by qRT-PCR. The relative gene expression level of HDGF was normalised to GAPDH. No statistical analysis was performed (melanocyte group is populated by only one type of cell line). (B) A375 and A2058 cells were infected with adenoviral vector at different MOI (100 or 200) then evaluated gene expression level of HDGF. (C) Representative images illustrating the effects of rHDGF (10 ng/mL) and Ad-HDGF shRNA (at 200 MOI) and Ad-GFP (at 200 MOI) on colony formation identified by crystal violet stains in A375 and A2058 cells. Quantitative measures of colony formation by counting the number of crystal violet positive cells. (D) Cells invaded through polycarbonate membrane (10 mm pore size) were stained with Giemsa. Representative photomicrographs of migrated cells through the Matrigel-coated filter were quantified in A375 and A2058 cells. All data are expressed as mean ± SEM from 3 experiments. *<i>P</i><0.05 compared to control, one-way ANOVA with post-doc analysis.</p