Tehran University Medical Journal

One of the main sources of a wide range of biological products as starting material is the human blood. These biological human plasma derived medicines play essential role in prevention and treatment of a variety of life threatening diseases. Mention to the starting material of these medicines which is blood or in another word human plasma, possibility of contamination by blood borne viruses cannot be omitted.
In recent years possibility of contamination by blood borne viruses such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) is an important concern. Nowadays most of developed countries the risk is minimum, although in developing countries it is still a challenge. Despite measures for human plasma biological derived medicines safety, such as donor selection, testing of donations, and polymerase chain reaction (PCR) testing on pooled plasma, still more actions are needed to inactivate or remove viruses such as HBV, HCV and HIV. During the process of manufacturing of biological human plasma medicines, there is several production steps which may contribute to viral reduction. These steps consist of precipitation by centrifugation, ethanol, polyethylene glycol, octanoic acid, or ammonium sulphate, chromatographic methods such as immunoaffinity chromatography or ion exchange chromatography, adsorption by aluminum hydroxide, and separation by filtration. All these steps are considered to be weakly effective as viral reduction treatment, and more effective viral inactivation methods are needed to be implemented in line of production of human plasma derived biological medicinal products. These safety measures included virus inactivation by different techniques such as acidic pH, solvent/detergent method, pasteurization and heat treatment, beta-propiolactone plus U/V and also virus removal by nanofiltration, which all these virus inactivation or virus removal methods before implementation in line of production of plasma derived biological medicines, should undergo for validation study.

Background: Thrombopoietin (TPO) is an important cytokine that has a critical role in regulating hematopoietic stem cells (HSCs) proliferation and megakaryocyte differentiation. Because of scares amount of this protein in human plasma, in many biotechnological centers around the world, recombinant production of this protein has been carried out. This study was aiming to gene cloning and expression of recombinant thrombopoietin.
Methods: This research is an experimental laboratory study carried out in Blood Transfusion Research Center, Tehran, Iran, from July 2016 to August 2017. At the beginning HepG2 cell line was cultured and RNA extraction was performed. Extracted RNA was used as template for cDNA synthesis and subsequently the synthesized cDNA was adopted to isolate TPO gene through polymerase chain reaction (PCR) reaction using designed primers. After isolating the TPO sequence from HepG2 cell line, the designated sequence was inserted into pET32 vectors. Recombinant plasmid was amplified by meriting from DH5α replicating system. The amplified plasmids were sequenced via chain termination method. Next step was transforming the recombinant plasmid into Rosetta-gami bacteria to express the recombinant protein. In order to induce protein expression, an appropriate amount of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to growth media, then bacterial lysate of expression host was prepared and assayed via polyacrylamide gel electrophoresis and western blotting test.
Results: After sequencing of recombinant plasmid, it was confirmed that TPO sequence has been successfully colonized in adopted vector. Subsequent to induction of recombinant protein, total cell protein analysis affirmed that recombinant protein has been expressed in its soluble form at cytoplasmic condition. Location of expected recombinant protein band on polyacrylamide gel and reaction of recombinant protein with His-tag monoclonal antibody at western blotting was asserting that expressed protein is the one of interest.
Conclusion: Rosetta-gami bacteria has capability of expressing recombinant thrombopoietin in its soluble form. By harnessing this method of recombinant protein expression, it would be possible to take advantage of high throughout bacterial expression system which would not produce inclusion body and its product doesn’t need further processing and refolding.

Background: Due to multiple roles of albumin in the body, injection of its medicinal product as one of the therapeutic or management strategies under conditions such as severe bleeding, burns, liver failure, and neonatal hemolytic diseases is on the physicians' agenda. Considering that albumin is the most abundant plasma protein, designing an appropriate method to purify it is highly important. There are several methods such as human plasma fractionation, chromatographic, or Salting-out methods for the isolation and purification of the human albumin. The present study investigates a direct and combined ion-exchange chromatography approach for purification of albumin from human plasma and compares the quality of the final products obtained by both ion-exchange chromatographic methods.
Methods: This study was carried out from January 2019 to October 2019 at the Blood Transfusion Research Center, High institute for research and education in transfusion medicine, affiliated with the Iranian Blood Transfusion Organization. For this study, 10 human plasma bags were randomly collected. After thawing, all 10 human plasma bags were pooled, and in order to separate cryo paste, it was centrifuged at 4000 g for 10 minutes at the temperature of 1 Centigrade degree. Then the obtained cryo poor plasma was used to purify the albumin protein by direct and combined methods of ion-exchange chromatography. The purity of the final products was compared by cellulose acetate electrophoresis and SDS-PAGE tests. The sample obtained by the combined approach was pasteurized and HPLC analysis was performed to investigate any polymer aggregates.