Separation And Process Automation Of The Plasma Proteins Form Cohn Franction IV Precipitation

The purpose of this study was to develope a process for isolation and purification of several pharmaceutical plasma proteins from the Cohn FranctionⅣprecipitates (CohnⅣprecipitates) which were often discarded in the manufacturing process of plasma products. The main components of CohnⅣprecipitates were Albumin (HSA), Transferrin (Tf),α1-antitrypsin (α1-AT), Antithrombin-Ⅲ(AT-Ⅲ) and Haptoglobin (Hp). A process scheme based on the combination of precipitation and multidimensional chromatography methods was designed. The CohnⅣprecipitates were prepared by precipitation methods, followed by multidimensional chromatography purification. Using this process scheme, we were able to purify the five proteins from Cohn IV precipitates conveniently and efficiently. Moreover, the separation could be achieved in an automatic manner.The pretreatment of CohnⅣprecipitates using polyethylene glycol (PEG) was firstly carried out, which involves dissolution of the precipitates in physiological saline solution, followed by centrifugation and filtration to remove the solid filtration aid. The protein solution was then precipitated by PEG to remove large molecular weight protein impurities, after which the CohnⅣprecipitate solution was obtained.Secondly, the preliminary chromatographic purification of CohnⅣprecipitate solution was researched, and the effects of the gel filtration chromatography (GF), hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEX) on purification were investigated. The results showed that preliminary separation of the precipitates could not be achieved using GF and IEX. However, the three fractions eluted from HIC contained less components and were more convenient for further purification. Therefore, Octyl Sepharose 4 Fast Flow (Octyl FF) was selected for the first chromatographic purification step. The stepwise elution using the gradient 20%,50% and 100% elution buffer resulted in three fractions. The major components in the three fractions were Tf,α1-AT and Hp, AT-Ⅲand HAS, respectively.The three fractions eluted from the Octyl column were then further purified by chromatographic methods. The first fraction was loaded onto a DEAE Sepharose Fast Flow (DEAE FF) column and Tf was eluted in a high purity, with a recovery of 74%. For the second fraction, DEAE FF column was used andα1-AT and Hp containing fractions were obtained. The Hp fraction was then loaded onto a GF column and Hp was collected in a high purity. The recovery ofα1-AT and Hp were 62% and 40%, respectively. The third fraction was loaded onto the Heparin Sepharose 6 Fast Flow (Heparin FF) column and AT-Ⅲand HSA were collected in high purities. The recovery of AT-Ⅲand HAS were 65% and 87%, respectively.Finally, the isolation and purification of pharmaceutical plasma proteins from the CohnⅣprecipitates was achieved on a High-throughput Integrated Column Chromatography System, and the five targeted proteins were obtained in high purity. The recovery of HSA, AT-Ⅲ,Tf,α1-AT and Hp were 95%,70%,80%,65% and 50%, respectively. Compared with the manual operation, the recovery was increased and the separation time was 5 times shorter. This system was joined by pipelines and could achieve automatic control of the whole purification process, which can save manpower, material, and avoid contamination in the operation.