Filteration method for cell produced antiviral substances

• US 4,420,398 A
• Filed: 08/13/1981
• Issued: 12/13/1983
• Est. Priority Date: 08/13/1981
• Status: Expired due to Fees

• Current Assignee: American National Red Cross
• Original Assignee: American National Red Cross
• Inventors: Castino, Franco
• Primary Examiner(s): Sadowski, David R.
• Application Number:
  US06/292,582
• Time in Patent Office: 852 Days
• Field of Search: 210/641, 210/645, 210/648, 210/650, 210/651, 210/774, 210/779, 210/790, 210/805, 210/806, 210/181, 210/182, 210/195.2, 210/257.2, 210/259, 210/295, 210/321, 210/335 , 210/433.2, 210/927, 435/68, 435/240, 435/241, 435/803, 435/811, 424/85
• US Class Current: 210/641
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/56 IFN-alpha
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 47/10 Separation or concentration of fermentation products bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02
• CPC Class Codes: C12M 47/12 Purification C12M47/04 takes precedence
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/811 Interferon

Abstract

This invention affords a filtration method and apparatus for extracting cell produced antiviral substances (CPAS) from a production broth using cross-flow membrane filtration. The broth is perfused into a first filtration cell and caused to cross-flow across an ultrafiltration membrane in the cell, permitting a first filtrate to pass through the membrane consisting essentially of CPAS and some molecular species and retaining in a first ex-filtrate all remaining portions of the broth.

Claims

1. A separation method for extracting Cell-Produced-Antiviral-Substances (CPAS) from a production broth containing the living cells which produce the CPAS consisting essentially of:

subjecting said broth and said living cells to a non-dead-end membrane filtration wherein the broth and said living cells are perfused into a first filtration cell and caused to flow across an ultra-filtration membrane in said cell which membrane has a nominal molecular weight cut-off of from twice to about five times the maximum molecular weight of the CPAS,

permitting a first filtrate to pass through said membrane consisting essentially of CPAS and some molecular species,

retaining in a first retentate all remaining portions of said broth, and wherein a diafiltration process is afforded by a partially-closed cycle continuous or semicontinuous flow, in which said first filtrate removed from the broth is replaced with an equal volume of media, thus maintaining a constant suspension volume throughout the process, and said first retentate is cycled back to the broth.

2. The method of claim 1 wherein a dual module filtration process is afforded by perfusing the first filtrate containing the CPAS into a second filtration cell and causing it to flow across a second filtration membrane in said cell, which second membrane has a nominal molecular weight cut-off of about 75 percent or less of the minimum molecular weight of the CPAS and permits a second filtrate to pass through it, consisting essentially of all molecules smaller than the CPAS, which second filtrate is then discarded, the CPAS molecules being retained in the second retentate.

3. The method of claim 1 wherein a dual module diafiltration process is afforded by perfusing the first filtrae containing the CPAS into a second filtration cell and causing it to flow across a second filtration membrane in said cell, which second membrane permits a second filtrate to pass through it, consisting essentially of all molecules smaller than the CPAS, which second filtrate is then recycled to the broth together with the first filtrate, maintaining an approximately constant suspension volume in a partially-closed cycle continuous or semicontinuous flow process.

4. The method of claims 2 or 3 wherein from 1 to 3 additional filtration cells are incorporated within the cycle whereby the CPAS of the second retentate is further purified by the filtration removal of molecular species and other impurities, which molecular species and other impurities are recycled to the broth.

5. The method of claims 2 or 3 wherein the production broth contains genetically engineered animal cells capable of producing a CPAS or hybridomas capable of producing a monoclonal antibody CPAS.

6. The method of claim 5 wherein the CPAS is one of the group consisting of human interferons, non-human animal interferons, lymphokines, or monoclonal antibodies.

7. The method of claims 2 or 3 wherein the production broth contains a mixture of a naturally occurring animal cell suspension and a CPAS inducer.

8. The method of claim 7 wherein the CPAS is at least one the group consisting of human interferons, non-human animal interferons, and lymphokines.

9. The method of claim 8 wherein:

the cell suspension consists essentially of human leukocyte cells from plasma,

the CPAS inducer is a virus, and

the CPAS produced is alpha-type human interferon (HuIFN-α

) having a molecular weight range of about 15,000 daltons to about 21,000 daltons.

10. The method of claim 8 wherein:

the cell suspension consists essentially of human fibroblast cells,

the CPAS inducer is a virus, and

the CPAS produced is beta-type human interferon (HuIFN-β

) having a molecular weight of about 24,000 daltons.

11. The method of claim 8 wherein:

the cell suspension consists essentially of human leukocyte cells from plasma,

the CPAS inducer is a mitogen, and

the CPAS produced is gamma-type human interferon (HuIFN/γ

) having a molecular weight range of about 40,000 daltons to about 75,000 daltons.

12. A dual module separation apparatus for extracting purified Cell-Produced-Antiviral-Substances (CPAS) from a CPAS production broth containing living cells which produce said CPAS consisting essentially of:

a source of production broth containing said living cells;

first non-dead-end filtration cell means, conduit means flowingly connecting said source of production broth containing said living cells to said first non-dead-end filtration cells means for flowing said broth and living cells across a filtration membrane for separating CPAS and all smaller molecules in a filtrate and recycling a retentate back to said source of production broth containing said living cells, which membrane has a nominal molecular weight cut-off of from twice to about five times the maximum molecular weight of said CPAS;

CPAS reservoir means for receiving and storing said CPAS;

conduit means flowingly connecting said first non-dead-end filtration cell means to said CPAS reservoir means for conducting said filtrate to said CPAS reservoir means;

second filtration cell means for separating molecules smaller than CPAS into a second filtrate and a second retentate containing CPAS, said second filtration cell means having a filtration membrane with a nominal molecular weight cut-off of about 75 percent or less of the minimum molecular weight of the CPAS;

and

conduit means flowingly connecting said second filtration cell means with said CPAS reservoir for conducting fluid from said CPAS reservoir to said second filtration cell means and for recycling said second retentate back to the CPAS reservoir.

13. The apparatus of claim 12 further comprising:

a production broth reservoir containing a CPAS;

a first non-dead-end conduit leading from said production broth reservoir to a first filtration cell;

said first filtration cell having a retentate chamber receiving the production broth from the first conduit, a second conduit leading from the retentate chamber back to the production broth reservoir, a filtrate chamber separated from the retentate chamber by a filtration membrane so that a perfusate into the cell flows across said membrane, which is sized to permit the passage of CPAS and all smaller or equal sized molecules;

a third conduit leading from said filtrate chamber to a CPAS reservoir;

a fourth conduit leading from said CPAS reservoir to a second filtration cell;

said second filtration cell having a second retentate chamber receiving the contents of the CPAS reservoir from the fourth conduit, a fifth conduit leading from the second retentate chamber back to the CPAS reservoir, a second filtrate chamber separated from the second retentate chamber by a second filtration membrane sized to permit the passage of all molecules smaller than the CPAS;

a sixth conduit leading from the second filtrate chamber to a waste disposal means;

means for adding production broth replacement ingredients to the production broth reservoir at a rate equal to that of the CPAS and smaller molecules removal; and

pumping means for maintaining flow through the conduits and throughout the apparatus at about a constant pressure or flow rate.

14. The apparatus of claim 12 or 13 wherein the filtrate from the second filtration cell is recycled by a seventh conduit, back to the production broth source.

15. The apparatus of claim 14 wherein a diafiltration circuit is included in a conduit between the CPAS reservoir and the second filtration cell.

16. The apparatus of claim 14 wherein cooling means are located between the first filtration cell and the CPAS reservoir, and heating means are located between the second filtration cell and the production broth source.

17. The apparatus of claim 12 or 13 wherein a dialysis circuit is included in a conduit between the CPAS reservoir and the second filtration cell, to remove contaminant salts.

18. The apparatus of claim 12 or 13 wherein the CPAS production broth consists essentially of CPAS producing cells CPAS inducer, culture media and culture adjuvants.

19. The apparatus of claim 12 or 13 wherein the CPAS producing cells consist essentially of human leucocytes, the CPAS inducer is a virus, and the CPAS is alpha-type human interferon.

20. The apparatus of claim 12 or 13 wherein the CPAS producing cells consist essentially of human fibroblast cells, the CPAS inducer is a virus, and the CPAS is beta-type human interferon.

21. The apparatus of claim 12 or 13 wherein the CPAS producing cells consist essentially of human leucocyte cells from plasma, the CPAS inducer is a mitogen, and the CPAS in gamma-type human interferon.

22. The apparatus of claim 12 or 13 wherein the CPAS is a lymphokine or monoclonal anitbody.

23. The apparatus of claim 12 or 13 wherein the CPAS producing cells consist essentially of genetically-engineered bacteria or hybridomas.

24. The apparatus of claim 12 or 13 wherein cooling means are located between the first filtration cell and the CPAS reservoir.