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Heidebrecht, Hans-Jürgen; Kainz, Bernadette; Schopf, Roland; Godl, Klaus; Karcier, Zueleyha; Kulozik, Ulrich and Förster, Beatrix (2018): Isolation of biofunctional bovine immunoglobulin G from milk- and colostral whey with mixed-mode chromatography at lab and pilot scale. In: Journal of Chromatography A, Vol. 1562: pp. 59-68

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The aim of the present work was to develop a new scalable and cost-efficient process to isolate bovine immunoglobulin G from colostral whey with high purity and minimal loss of activity. The mixed mode material Mercapto-Ethyl-Pyridine-HypercelTm was identified appropriate for direct capture of immunoglobulin G. The binding mechanism is primarily based on hydrophobic interactions at physiological conditions. As compared to immunoglobulin G, all other low molecular whey proteins such as a-Lactalbumin or P-Lactoglobulin, except lactoperoxidase, are more hydrophilic and were therefore found in the flow-through fraction. In order to remove lactoperoxidase as an impurity the column was combined in series with a second mixed mode material (Capto(TM-) with N-benzoyl-homocysteine as ligand) using the same binding conditions. At pH 7.5 the carboxyl group of this ligand is negatively charged and can hence bind the positively charged lactoperoxidase, whose isoelectric point is at pH 9.6. After sample application, the columns were eluted separately. By combining the two columns it was possible to obtain immunoglobulin G with a purity of >96.1% and yield of 65-80%. The process development was carried out using 1 mL columns and upscaling was performed in three steps up to a column volume of 8800 mL for the Hypercel(TM) column and 3000 mL for the Capto(Tm)- column. At this scale it is possible to obtain 130-150g pure immunoglobulin G from 3 L colostrum within five hours, including the regeneration of both columns. Additionally, the impact of freeze-drying on the isolated immunoglobulin G was studied. The nativity of the freeze dried immunoglobulin was above 95%, which was proven by reversed phase liquid chromatography and validated by differential scanning calorimetry. The activity of immunoglobulin G was preserved over the isolation process and during drying as measured by enzyme-linked immunosorbent assay. In conclusion, by applying the proposed isolation process, it becomes feasible to obtain pure, active and stable imunnunoglobulin G at large scale.

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