Junting Jia

Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma

As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource.

Inactivation of viruses during a new manufacturing process of α2-macroglobulin from Cohn Fraction IV by dry-heat treatment.

α2‐Macroglobulin (α2‐M) has a curative effect on radiation injury. Virus transmission through plasma derivatives is still not risk‐free. Effect of dry heat on α2‐M activity and virus inactivation by dry heat in a new manufacturing process of α2‐M were studied.

Construction and characterization of an infectious replication competent clone of porcine endogenous retrovirus from Chinese miniature pigs

BackgroundXenotransplantation from animals has been considered to be a preferable approach to alleviate the shortage of human allografts. Pigs are the most suitable candidate because of the anatomical and physiological similarities shared with humans as well as ethical concerns. However, it may be associated with the risk of transmission of infectious porcine pathogens. Porcine endogenous retroviruses (PERVs) are of particular concern because they have been shown to infect human cells in vitro. To date, researches on the molecular characteristics and potential pathogenicity of PERV are still tenuous. In this report, an infectious replication competent clone of PERV from Wuzhishan pigs (WZSPs) in China was generated and characterized. This infectious clone will contribute to studies on PERV virology and control of PERV in xenotransplantation using Chinese miniature pigs.MethodsThe proviral DNA of PERV from WZSPs was amplified in two overlapping halves. Then the two fragments were isolated, subcloned and fused to generate pBluescriptαSK+-WZS-PERV recombinant clones. Screened with RT-PCR, a molecular clone of PERV designated as WZS-PERV(2) was selected. Its infectivity and replication competency were characterized in HEK293 cells by PCR, real-time fluorescent quantitative RT-PCR, western blot, indirect immunofluorescence assay as well as sequence analysis.ResultsThe ability of WZS-PERV(2) to infect human cells and produce infectious virions were shown after transfection of the clone into HEK293 cells and infection of PERV derived from this recombinant clone. The expression of Gag proteins were detected in HEK293 cells infected with the virus derived from the clone by the indirect immunofluorescence assay and western blot. The results of sequences analysis and comparison combined with the PCR based genotyping result demonstrated that the WZS-PERV(2) belonged to PERV-A subgroup. Compared with a previous proviral DNA clone of PERV (PERV-WZSP), G to A hypermutation occurred in the env gene of WZS-PERV(2) was found, whereas APOBEC proteins have the potential to inhibit the replication of a variety of retroviruses through a cDNA cytosine deamination mechanism, so we presumed these G to A hypermutation might be the contribution of porcine APOBEC3F.ConclusionsAltogether, an infectious replication competent clone of PERV from Chinese miniature pigs (WZSPs) termed WZS-PERV(2) was generated, characterized and sequenced.

Large‐scale purification of high purity α1‐antitrypsin from Cohn Fraction IV with virus inactivation by solvent/detergent and dry‐heat treatment

α1‐Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large‐scale purification of AAT from it. liquid chromatography‐electrospray ionization‐tandem mass spectrometry and high‐performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry‐heat. Virus inactivation by dry‐heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5 IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry‐heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1 H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry‐heat. The advantages of this process, including suitability for large‐scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.

Simultaneous detection and differentiation of human parvovirus B19 and human parvovirus 4 by an internally controlled multiplex quantitative real-time PCR

Human parvovirus B19 (B19V) and human parvovirus 4 (PARV4) are two parvoviruses known to infect humans and transmit through blood and plasma derived medicinal products (PDMPs). Inactivation of the two parvoviruses has proven to be difficult and nucleic acid testing (NAT) would be an efficient means to exclude viruses. In this study, an internally controlled multiplex quantitative real-time PCR (qPCR) assay for B19V and PARV4 simultaneous detection and quantification was established and evaluated. The optimized multiplex qPCR assay allowed for simultaneous detection of all of the genotypes (1-3) of B19V and PARV4, with equal limit of quantification (LOQ) of 5 copies/μL, rather than other blood-borne viruses. It had a wide dynamic range of reliable amplification linearity of at least 8 orders of magnitude. Low standard deviations (SD) of quantification cycle (Cq) values and low coefficients of variation (CV) of copy numbers for both B19V and PARV4 suggested a high level of repeatability and reproducibility for the multiplex qPCR assay. This multiplex qPCR assay can be served as a readily applicable approach to screen plasma units intended for further manufacturing into PDMPs to reduce the risk of parvoviruses infection by such products and may also be useful for the detection of B19V/PARV4 co-infection or co-existence.