RESUMEN
Commercial fractionation of human plasma into immunoglobulin- and albumin-rich fractions is often initiated with sequential cold ethanol-based precipitation methods, which have changed little over the past 70 years. The required low temperature (-4 to -8°C) and high concentrations of ethanol 8-40%) necessitate large-scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen-, immunoglobulin-, and albumin-rich fractions with 80-90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG-rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin-rich precipitate using a two-step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity.
Asunto(s)
Resinas Acrílicas/química , Albúminas/aislamiento & purificación , Fraccionamiento Químico/métodos , Inmunoglobulina G/aislamiento & purificación , Plasma/química , Albúminas/química , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Caprilatos/química , Precipitación Química , Cromatografía por Intercambio Iónico , Humanos , Inmunoglobulina G/químicaRESUMEN
The manufacture of human serum albumin by chromatographic procedures involves gel filtration chromatography as a final polishing step. Despite this step being essential to remove high molecular weight impurity proteins and thus ensure a stable and safe final product, it is relatively inefficient. This paper explores the use of hydrophobic charge induction chromatographic media, MEP HyperCel as an alternative to Sephacryl S200HR gel filtration for the polishing of human serum albumin derived by ion exchange chromatographic purification of Cohn Supernatant I. The use of MEP HyperCel results in a product with a higher purity than achieved with gel filtration and in a less time consuming manner and with potential resource savings. MEP HyperCel appears to have great potential for incorporation into downstream processes in the plasma fractionation industry as an efficient means of achieving polishing of intermediates or capture of proteins of interest.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Albúmina Sérica/aislamiento & purificación , Cromatografía por Intercambio Iónico/instrumentación , Contaminación de Medicamentos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Albúmina Sérica/análisis , Albúmina Sérica/química , Albúmina Sérica/normasRESUMEN
Human serum albumin is a well tolerated therapeutic for the treatment of hypovolemia. Despite all commercial human albumin preparations being derived from plasma, these products can have a highly variable colour. Albumin samples derived from ethanol precipitation and chromatographic fractionation procedures were evaluated for bilirubin and biliverdin levels and by spectrophotometry. It was shown that albumin derived from a chromatographic process, which had a bilirubin:albumin ratio similar to that observed in plasma, had a vibrant yellow appearance. The albumin derived from ethanol precipitation had undetectable levels of bilirubin, and the amber colour of this product was attributed mainly to residual haem. The presence of bilirubin during pasteurisation led to oxidation to biliverdin, with a resultant colour change from yellow to yellow/green. Given that the antioxidant properties of bilirubin are well established, it is possible that bilirubin helps protect albumin from oxidation during the pasteurisation step.
Asunto(s)
Color , Composición de Medicamentos/métodos , Contaminación de Medicamentos , Albúmina Sérica/síntesis química , Bilirrubina/análisis , Bilirrubina/aislamiento & purificación , Biliverdina/análisis , Biliverdina/aislamiento & purificación , Color/normas , Composición de Medicamentos/efectos adversos , Contaminación de Medicamentos/prevención & control , Calor/efectos adversos , Humanos , Hierro/análisis , Hierro/aislamiento & purificación , Luz/efectos adversos , Pigmentos Biológicos/análisis , Pigmentos Biológicos/aislamiento & purificación , Pigmentos Biológicos/farmacología , Albúmina Sérica/análisis , Albúmina Sérica/química , Albúmina Sérica/efectos de la radiación , Esterilización/métodosRESUMEN
This study evaluated the feasibility of substituting expanded bed adsorption (EBA) chromatography for an existing chromatographic purification process for the isolation of prothrombin complex concentrate (PCC) from Cohn Supernatant I. The EBA chromatography (Streamline) resins were compared to the current DEAE-cellulose resin for the extraction of PCC from Cohn SNI. EBA chromatography resins efficiently bound PCC from Cohn SNI at a significantly higher flow rate of up to 300 cm/h compared to 30 cm/h for the current DEAE-cellulose process. Composition and yield of the recovered PCC reflected the elution conditions used. The results indicate that EBA chromatography could be used to efficiently produce PCC comparable to existing products.
Asunto(s)
Factores de Coagulación Sanguínea/aislamiento & purificación , Proteínas Sanguíneas/química , Cromatografía/métodos , Resinas de Intercambio Aniónico/farmacología , Técnicas de Laboratorio Clínico , Industria Farmacéutica/métodos , Humanos , Protrombina/aislamiento & purificación , Albúmina Sérica Bovina/químicaRESUMEN
The present paper describes an anion-exchange chromatography method to separate iron-free apo-Tf (apo-transferrin) from albumin and IgG in Cohn supernatant I. The method uses DEAE-fast flow Sepharose chromatography along with optimized protein concentration (5%, w/v) and column operation conditions (40 g/l, conductivity <1.0 mS/cm) to resolve apo-Tf from IgG and albumin. The single step purifies apo-Tf to >90% and provides an efficient means to obtain commercial quantities of the protein.
Asunto(s)
Cromatografía por Intercambio Iónico , Transferrina/aislamiento & purificación , Fraccionamiento Químico , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Estudios de Factibilidad , Humanos , Inmunoglobulina G/aislamiento & purificación , Albúmina Sérica/aislamiento & purificaciónRESUMEN
There are many proteins that can multi-task. Transferrin, widely known as an iron-binding protein, is one such example of a multi-tasking protein. In this review, the multiple biological actions of transferrin, including its growth and cytoprotective activities, are discussed with the view of highlighting the potential therapeutic applications of this protein.