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

• This is the first study that Cohn Fraction IV was used to purify α2-macroglobulin, which makes better use of human plasma.
• Yield of 45% and purity of 95% were achieved simultaneously regardless of haptoglobin type.
• Simple process of salting-out method and immobilized metal affinity chromatography facilitate pilot scale preparation.
• α2-M could be readily inactivated on the condition of temperature above 50 °C, pH above 9.0 or below 4.0, and the existence of methylamine.
• This is the first study that Cohn Fraction IV was used to purify α2-macroglobulin, which makes better use of human plasma.
• Yield of 45% and purity of 95% were achieved simultaneously regardless of haptoglobin type.
• Simple process of salting-out method and immobilized metal affinity chromatography facilitate pilot scale preparation.
• α2-M could be readily inactivated on the condition of temperature above 50 °C, pH above 9.0 or below 4.0, and the existence of methylamine.

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.