Methods for preparation of highly purified, gamma globulins free of hepatitis-B-virus infectivity

Claim 1

A method for removing hepatitis B infectivity from a gamma globulin containing body fluid comprising the steps of: (a) providing the gamma globulin containing body fluid desired to be purified; (b) applying the body fluid to first and second column means in series, said first column means containing an effective amount of a rigid resin consisting of DEAE-Sepharose CL-6B, DEAE-Sepharose CL-6B (Fast Flow) or DEAE Bio-Gel (High Capacity); and said second column means containing an effective amount of a non-rigid resin consisting of DEAE-Sephadex or QAE-Sephadex; (c) eluting the body fluid from said first column means with a buffer adjusted to a pH of at least 7.0, said buffer consisting of approximately 0.02 M phosphate buffer if the resin selected for said second column means is QAE-Sephadex, or said buffer consisting of approximately 0.04 M Tris buffer, approximately 0.05 M Imidazole buffer, or approximately 0.035 sodium phosphate [Na 2 HPO 4 ]-citric acid buffer, if the resin selected for said second column means is DEAE-Sephadex or QAE-Sephadex; (d) monitoring the effluent from said first column means for the presence of protein; (e) collecting, responsive to monitoring, the protein containing effluent; (f) applying the collected protein containing effluent from said first column means to said second column means; (g) eluting the fluid from said second column means with one of said buffers which may be utilized in said first column means, said buffer being adjusted to a pH of at least 7.0, the buffers used in the first and second column means respectively, being the same or diferent; and (h) collecting the effluent from said second column means whereby any hepatitis-B-virus infectivity is now removed.

Claim 2

A method for removing hepatitis B infectivity from a gamma globulin containing body fluid comprising the steps of: (a) providing the gamma globulin containing body fluid desired to be purified; (b) applying the body fluid to first and second column means in series, said first column means containing an effective amount of a rigid resin consisting of DEAE-Sepharose CL-6B, DEAE-Sepharose CL-6B (Fast Flow) or DEAE Bio-Gel (High Capacity); and said second column means containing an effective amount of a non-rigid resin consisting of DEAE-Sephadex or QAE-Sephadex; (c) eluting the body fluid from said first column means with a buffer adjusted to a pH of at least 7.0, said buffer consisting of approximately 0.02 M phosphate buffer if the resin selected for said second column means is QAE-Sephadex, or said buffer consisting of approximately 0.04 M Tris buffer, approximately 0.05 M Imidazole buffer, or approximately 0.035 sodium phosphate [Na 2 HPO 4 ]-citric acid buffer, if the resin selected for said second column means is DEAE-Sephadex or QAE-Sephadex; (d) monitoring the effluent from said first column means for the presence of protein; (e) collecting, responsive to monitoring, the protein containing effluent; (f) applying the collected protein containing effluent from said first column means to said second column means; (g) eluting the fluid from said second column means with one of said buffers which may be utilized in said first column means, said buffer being adjusted to a pH of at least 7.0, the buffers used in the first and second column means respectively, being the same or diferent; and (h) collecting the effluent from said second column means whereby any hepatitis-B-virus infectivity is now removed.