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Validation of Recovery and Purification Processes
Published in James Agalloco, Phil DeSantis, Anthony Grilli, Anthony Pavell, Handbook of Validation in Pharmaceutical Processes, 2021
In another example of a study to validate DNA clearance during purification of a recombinant protein, 21 consecutive purification cycles were performed using three different anion exchange chromatography media.49 Radio-labeled DNA was spiked into the column load before each cycle and after every five cycles, and the clearance factor for removal of all DNA and DNA of greater than 50 base pairs was determined. For each chromatography media, the clearance factor was consistent throughout the validation study. The average clearance factor for two Sepharose Fast Flow anion exchangers (Q Sepharose and DEAE Sepharose) of approximately 1.5 million was obtained. For DE-52 Cellulose (Whatman, Clifton, NJ) the clearance factor was approximately half that of the Sepharose exchangers or 0.7 million.35 Each of these validation studies demonstrated that a final concentration of DNA of less than 100 pg per dose of protein could be reproducibly achieved, which was the limit at the time of these studies.
Fish Growth Hormones: Production and Application of Recombinant Hormones
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
The inclusion bodies are solubilized in buffer A: 10 mM Tris-HCl buffer (pH 8.0) containing 7 M urea. A nonionic denaturing agent, urea, was used to perform the following ion-exchange chromatography. Most rsGH I in the inclusion bodies was solubilized in reduced form (see Fig. 5, lane b). The supernatant was applied onto a DEAE-Sepharose fast flow column (20-cm ID x 30 cm, Pharmacia), equilibrated with buffer A. Recombinant sGH I was eluted with a gradient of NaCl from 0 M (20 L) to 0.33 M (20 L) in buffer A (Fig. 6). As shown in Figure 7, rsGH I monomer was separated from rsGH I aggregates and from most impurities. Fractions 14-20 were pooled and subjected to a subsequent purification process.
Process Affinity Chromatography
Published in Juan A. Asenjo, Separation Processes in Biotechnology, 2020
Dye-ligand chromatography is probably the type of pseudoaffinity chromatography most appropriate for large-scale applications (Scawen and Atkinson, 1987; Clonis, 1988b). The initial development of a large-scale process often involves an analytical-scale screen of many immobilized dye ligands. Parameters to consider include adsorbent capacity, fold of purification, ease of elution, and yield obtained. After narrowing the dyed adsorbents to a limited number of “possible, more detailed studies—for example, to determine the optimal ligand concentration, pH, ionic strength and so on—performed (Bruton and Atkinson, 1979; Clonis and Lowe, 1981; Scawen and Atkinson, 1987). An interesting but not typical example is the purification of malate dehydrogenase (MDH) and hydroxybutyrate dehydrogenase (HbDH) from the same microorganism, Rhodopseudomonas spheroides (1 kg of paste). These enzymes were purified in two consecutive chromatographic steps using the dye ligands Procion Red H-3B and Procion Blue MX-4GD (Scawen et al., 1982). First, on the red column (1.8 L), HbDH was eluted with KCl, while MDH could only be desorbed with NADH in the presence of KCl. Then, on the blue column (0.4 L), HbDH was eluted by NADH in the presence of salt, whereas MDH was desorbed in salt alone. This combination of two dye ligands functioning in a complementary fashion enables the two enzymes to be purified in a short time to homogeneity with yields up to nearly 80%. Another example where a dye ligand has been used in large-scale work is the purification of glycerokinase from 1 kg of Bacillus stearothermophilus paste (Hammond et al., 1986). This process involves cell homogenization in a Manton-Gaulin ion-exchange chromatography on a DEAE-Sepharose CL-6B column (2 L), and dye-ligand chromatography on a Procion Blue MX-3G-Sepharose 4B column (0.6 L). This method produces 0.7 g of homogeneous enzyme, of 4.9-fold purification, with a yield of 44%.
Biodegradation of cyanide to ammonia and carbon dioxide by an industrially valuable enzyme from the newly isolated Enterobacter zs
Published in Journal of Environmental Science and Health, Part A, 2021
Zohre Javaheri Safa, Arta Olya, Mohammadreza Zamani, Mostafa Motalebi, Rahimeh Khalili, Kamahldin Haghbeen, Saeed Aminzadeh
The cyanide degrading enzyme was purified from Enterobacter cell-free extract. It was then separated from the crude extract on DEAE sepharose anion-exchange chromatography. The enzyme was obtained from the unbound fraction, and its absorbance at 280 nm was measured 1.8 (Fig. 1). Enzyme activity was assessed by performing ammonia assay. This enzyme could degrade cyanide in 474.5 mg.L−1 in 60 min. Table 2 recapitulates the purification steps. SDS-PAGE demonstrated that the purified protein band was somewhere in the region of 20 kDa (Fig. 2).