Natural interferon production has traditionally involved ammonium chloride treatment of buffy coats to lyse the red blood cells and to isolate the leukocytes, followed by viral stimulation of leukocytes with subsequent large scale harvesting of culture medium. The interferons are then isolated by various precipitation, adsorption, or immuno-affinity techniques.

Concentration of Interferon from Luekocyte Culture:

Traditionally, luekocytes have been collected as whole blood and stored in impermeable plastic bags at about 4 C-25C until processed into plasma and red blood cells. The white blood cell layer (buffy coat) is generally discarded as a side product which can be collected and treated with ammonium chloride to lyse the contaminating red blood cells. The remaining leukocytes are then cultured in media containing a serum and activated with a viral inducer to produce interferon. Morris (US6,350,589) disclosed a process where whole blood is collected and centrifuged to produce a buffy coat. Collection of the buffy coat is done with a peristaltic pump and a specially designed manifold that is much gentler than a vacuum pump. An initial wash with PBS serves to remove both platelets and some serum contaminants. After centrifugation, the supernatant is removed with a peristaltic pump and specially designed aspirators. The cells are gradually brought to isotnoic osmolarity with PBS washes. 

Subsequent Purification Techniques

Affinity Purification Techniques:

–Antibody affinity – RPHPLC: US 4,551,271 discloses passing a partically purified preparation through an antibody affinity column and a reversed phase high performance liquid chromatographic column. 

Controlled Pore Glass (CPG):

Billiau (Antimicrobial Agents and Chemotherapy, July 1979, p. 49-55) disclsoes purification of human fibroblast interferon by absorption of F-inteferon on CPG beads at neutral pH and more or less selective relase at pH about 2.0. 

–CPG – Zinc-Chelate Chromatography:

Heine (Methods in Enzymology, 78, pp. 448-456, 1981) discloses purificaiton of crude human fiblast interferon by CPG adsorption and then a stecond step of zinc-chelate chromatographic purificaiton. 

Hydroxyapatite (HA) chromatography: Morris (US6,350,589) discloses taking a crude interferon (e.g., such as a cation exchange capture) and applying it to a HAC column. 

Particular types of Interferons

INF-alpha: Bodo (US 5196323) discloses a process for purification of rrecombinant  IFN-alpha by tandem chromatography comprising separation on a cellulose column followed by an anti-alpha-interferon monoclonal antibody affinity column, then subjecting the purifed material to isolectric precipitation of impurities at about pH 4.0 to about pH 4,8 and then chromatography on a high performance cation exchange solumn using a volatile buffer.