For virus removal from plasma proteins by filtration see “filtration” and “particular components”

Taking into consideration how many blood donations are necessary for the production of IVIG, the prevention of the transmission of viruses is a key aspect in the development of such products. The state of hte art viral reduction or inactivation steps usually integrate into the purificaiton process the use o chemical agents as for instance during solvent/detergent (S/D) treatment, extreme physical conditions, such as low pH incubation or high termpature during pastuerization, ultraviolet light irradiation or exploiting the sieze difference to the target protein as for example in nanofiltration (Teschner “A new liquid, intravenous immunoglobulin product (IGIV 10%) highly purified by a state of the art process” VoxSanguinis 2007, 92, 42-55).

Kriel teaches 3 dedicated virus reduction steps (1) 3 component solvent detergent treatment, (2) a 35 nannofiltration step and (3) a prolonged incubation period at low pH and elvevated temperature (Kriel, J. Allergy Immunol., Februayr 2004, pS128 abstracts).

Treatments used Generally for Viral Inactivation

Low pH

Most proteins are damaged by exposure to the acidic conditions needed to kill viruses. For example, few viruses are killed at pH 5-5.5, a condition known to inactive factor VIII. Immune globulin solutions are an exception. Various studies have shown that low pH, such as pH 4 used in the preparation of immunogloublins, inactivates several enveloped viruses. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

Low pH treatemtns at 37C for 20 h or more, initially intorduced to reduce anticomplementary activity, were later identified to inactivate >4 logs of several enveloped viruses including HIV and various model virruses. Incubation of IVIG at e.g., pH 4.25 for 3 weeks at 21C inactivates enveloped virsues such as HCV. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28.

Solvent/detergment (S/D) treatment

Organic solvent/detergent mixtures disrupt the lipid membrane of enveloped viruses. Once dsrupted, the virus can no longer bind to and infect cells. Non-enveloped viruses are not inactivated. The conditions typically used are 0.3% tri_n-butyl) phosphate (TNBP) and 1% nonionic detergent, either Tween 80 or Triton X-100, at 24C for a minimum of 4 hours with Triton X-100, or 6 hours with Tween 80. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

S/D treatment has been used for viral inactivation of IVIG. The prcoess involves an incubation of a IVIG liquid intermediate in the presence of a solvent (tri-n-butylphosphate) adn generally one, sometimes two detergents (polysorbate 80, Triton X-100 and less frequently, sodium cholate). In most processes the incbuation lasts at least 1-6 h and is carried out at 20-35 C. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

–solvent/detergent treatment, 35-nm nanofiltration and low pH/elevated termpature incubation (Poelsler “A new liquid intravenous immunoglobulin with three dedicated virus reduction steps: virus and prion reduction capacity” Vax Sanguinis 2007). 

–(7655233, S/D treatment after fractionation and nanofiltration).

high temperature during pasteurization:

A problem with inactivating viruses with gamma globulins, particularly IgG, is that they have a poor heat stability. In this respect, stabilizers have been used. Hirao (EP0196761A2) discloses that the thermal stability of gamma globulin against heat treatment is markedly improved when at least one stabilizer such as a monosaccharide (glucose, mannose, galactose and fructose), a disaccharide (sucrose, maltose, lactose) or a sugar alcohol(mannitol, sorbitol, xylitol)  is added to an aqueous solution containing the gamma globulin prior to or at the time of the heat treatment. Thermal stability of the gama-globulin is further enhanced when at least one member slected from a neutral amino acid (glycine, alanine, valine, leucine, isoleucine), a neutral inorganic acid salt (halide of alkali metals or alkaline earch metals such as sodium chloride, potassium chloride and magnesium chloride), a surface active agent (nonionic surface active agents such as alkylphenyl-polyoxyethylene, anionic surrace active agents such as bile acid salts and cationic surface active agents such as benzalkonium chloride and polyhdric alcohols) and an organic carboxylic acid  (hydrocarbon residue such as an alkyl group or an aryle group (such as a phenyl group) and a carboxyl substitute attached), preferably about 3-15 carbon atoms,  is added to the solution in addition to the stabilizer. The kind of salts of the oranic carboxylic acids include alkali metal salsts such as sodium salts and potassium salts and alkaline earth metal satls such as calcium salts. Examples of the organic acid salts include alkali metal salts (sodium or potassium salt) of propanoic, butanoic, pentanoic, caprylic, caproic, malonic, succinic, tglutaric, adipic, citric and mandelic acid.

It is particularly suitable if prior to a virus inactivation treatment, the plasma protein recovered is further subjected to a dialyses against a medium with a low salt content (e.g., water) which may provide an additional stabilizing effect for the plasma protein because of the present of the mono- or dicarboxylate. Teschner (US8,709,492B2). 

Chen (US6,338,849) discloses process for preparing IVIG comprising dissolving Cohn’s component I in ice water, adjusting pH to between 3.5 and 5, filtering with an UF to remove alcohol and salt, sterilizing the immunoglobulin concentration at a temeprature between 59 and 6C for 10 hours and then filtering with an UF. 

Feldman (4,876,241) discloses inactivating pathogens in proteinaceous biological products by adding primary stabilizers selected from the group consisting of sugars and sugar alcohols and one or more secondary stabilizers selected form the group consisting of sodium acetate, potassium acetate, lithium acetate, magnesium acetate, ammonium acetate, barium acetate, sodium sulfate, ammonium sulfate, lithium sulfate, potassium sulfate and magnesium sulfate and subsecting the mixture ot a pathogen inactivating prcoess 

Precipitation: 

A very effective removal of viruses is accomplished by precipitation, especially where the precipitate is a waste fraction. Thus, the plasma protiens like albumin and IgG which are dervied after several subsequent ethanol precipitation steps are regarded as very safe.

Precipitation with ethanol is the single most widely used plasma fractionation tool worldwide. In addition to its use as a precipitant, ethanol is also a disinfectant. Unfortunately, it acts as a disinfectant mostly at room temperature or above, whereas plasma fractionation is carred out at a low temeprature to avoid protein denaturation. The contribution of ethanol ot viral safety through inactivation is, therefore, marginal at best. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

Precipitation/filtration/diafiltration/dialysis: Starting Cohn Fraction II paste: is typically about 95% IgG and is comprised of the 4 IgG subtypes. Precipitation: Dissolve in a cold purified alcohol solution and impurities removed via precipitation and filtration. The IgG suspension can be dialyzed or diafiltrated (e.g., using ultrafiltration membranes have a nomimal MW of less than or equal to 100k daltons to remove the alchol. The solution can be concentrated or diluted to obtained the desired protein concentration and further purified (US20090148463).

Filtration:

Barrett (WO99/43362) discloses a method for eliminating pathogens from a solution containing plasma proteins by filtration. The solution is incubated in the presence of an inorganic, particulate, surfactant detergent and filtered in a deep filter and a clear solution is obtained. 

Van Holten (US6,096,872) discloses method for making virus free immunoglobulin formulation by missing an immunoglobuiln fraction isolated form human plasma with a high ionic strengh buffer containing a non-ionic detergent, performing nanofiltration using less than about 30 nm nanofile and collecting permeate of the nanofiltration. 

–Nanofiltration Filters: with nomimal poor sizes in the namometer range are well established tools for enchacing virus safety margins of plasma derived products, (Kreil, Transfusion, 46, 2006). Viral removal cn be chieved by nonfiltration where purified immunoglobulin solutions are passed through commerical filters of a controlled pore-size (typically 15, 20 or 35 nm) and defined design to retain viruses based on steric exclusion. The process has been shown to be robust and simple and contributes to the removal of both enveloped and non-enveloped viruses. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Irradiation with ultraviolet light (UVC):

ultraviolet irradiation, typicaly at a WL of 254 nm targets nucleic acid, thus a wide variety of viruses are inactiated, irrespective of the nature of their envelope. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

Caprylic aicd (Caprylate) (also known as octanoic acid): (see also fractionation -fatty acids)

Besides its role in IgG purification, caprylic acid in the non-inoniced form also acts as an agent that can robustly inactive lipid-enveloped viruses within a few minutes when used at pH < 6 and a concentraiton of >3.7 g/l. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Debart (US 15/27655, published as US 20170198009) disclsoses a method of preapring a solution of immunogloublin from an initial solution such as a fraction II+III in the presence of a polymer of glyocol such as polyethylene glycol (PEG) by adding caprylic acid to a concentration of 9-15 mM, adjusting the pH, incubating the solution for a time and temperature to inactviate enveloped virsues and then UF/DF. The us of cparylate at a lower concentraiton than described in the prior art and in the prresence of at least one polyether or polymer of glycol is disclosed as avoiding aggregates which forms as a result of cpaprylate at the concentraiton and pH (e.g., pH 5-5.2) in the prior art 

Lebing (US 5,886,154) teaches the use of caprylate for viral inactivation.

Seng (US4,939.176) reports a process for inactivating viruses by contacting solutions with caprylic acids with pH 4-8. 

Combination of viral reduction treatments: 

The newest generations of IVIG have unprecedented margin of viral saftety afforded by a combination of complementary dedicated viral reduction treatments, such as low pH treatment/nanofiltration, caprylic acid/low pH treatment, pasteurization/nanofiltration, S/D.nanofiltration/low pH treatment. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Anion Exchange with Viral Inactivation Steps

AEX-Viral Inactivation-CEX:  Burnouf-Radosevich (US 6,069,236) disclose a process for preparing immunoglobulin G from plasma which includes an anion exchange gel, a viral inactivation treatment and a cation exchange gel.

Precipitation-AEX-CEX-Virus inactivation-AEX-CEX: Laursen (6,281,336; WO99/64462) disclose a process for purifying IgG from crude plasma by adding a water soluble non-denaturating protein precipitant to preipitate a high proportion of non-immungolublin G proteins, recovering the clarified IgG supernatnat and applying it to an AEX and subsequently a CEX, eluting the IgG from the CEX and performing DF/UF, adding a virucidal amoung of virus inactivating agent, applying the IgG to AEX and subsequently to CEX, eluting the IgG from the CEX and performing DF/UF. 

precipitation (caprylate) – viral inactivation (sodium caprylate) -AEX:  Lebing, (US 5,886,154; EP0893450B1), disclose purification of antibodies from human plasma and other sources by suspending the antibodies at pH 3.8-4.5 followed by addition of caprylate acid and a pH shift to pH 5.0 to 5.2 to precipitate of contaminating proteins, lipids and caprylate forms is removed, while the majority of the antibodies remain in solution. Sodium caprylate is again added to a final concentration of not less than about 15 mM and incubated under conditions to reduce active virus (e.g., 1 hour at 25C). A precipitate (Mianly caprylate) is removed and the clear solution is diluted with water to reduce ionic strenght. Anion exchange is then used to obtain pure antibody prepration. (Lebing, Vox Sang 2003 (84):193-201) also disclose that caprylate is added to precipitate non-IgG proteins and to inactive enveloped viruses) (US20070244305, caprylic acid treatment, anion exchange, filtering).

Precipitation-AEX-UF-Virus Inactivation: (Ristol Debart (EP1225180B1)) discloses starting with a precipitate rich IgG obtained by ethanolic fractionation of human plasma, suspending the precipitate in an aqueous solution which contains a carbohydrate, preferably a sugar alcohol such as sorbiotol, precipitating the IgG accompanying globulins with PEG, filtration of the precipitate, bringing the filtrate to a pH of 5.7-6.3 and subjecting it to ion excahnge with anionic ligand, preferably those of DEAE. The liquid effluent is ultrafiltrated to reduce PEG contact and obtain a suitable concentration of protein to carry out subsequent steps of viral inactivation. 

AEX-Pastueurization-DF/UF: Aghaie (Human Antibodies 19 (2010) pp. 1-6) discloses redisolving Fraction II paste with glycine buffer and performing AEX on CM sepharose fast flow (FF). The eluate was then dia-ultrafiltered and sorbitol and glycine were added as stabilizers and the solution was pasteurized in water bath of 60C for 10 hours. The pastuerized solution was then filtered and then DF/UF. 

Cation Exchange with Viral Inactivation Steps

CEX-Virus Inactivation: Andersson (US2003/0143222; WO/2001/072844) discloses a method for producing IgG from plasma by removal of albumin, then adsorbing IgG to a CEX , collecting the adsorbed IgG fraction, adjusting pH to 4 (permits virus inactvation to be carried out around 30C) and virus inactivation of the Ig using chemicals such as a solvent/detergent (S/D) solution at 30C for at least 4 hours

Precipitation-Virus Inactivation-CEX-AEX: Sarno (EP0440483A2) discloses a multip step process for purifying an immune serum globuli fraction from crude plasma comprising precipitating non-serum globulin proteins from an aqueous supension of the crude plasma protein fraction, vrus inactivation, followed by absorbing the immune serum globulins onto a cation exchange followed by anion exchange to absort non-serum globulin contaminants.

CEX-Virus Inactivation-AEX: Andersson (US6835379) discloses a method for producing IgG from plams by removal of albumin resulting in an IgG fraction, absorbing Ig to a cation exchanger and collecting the absorbed IgG fraction, virus inactivation and and purifying plasma fraction obtained after remo

removal of euglobulins and albumin on an anion exchanger.  

CEX (strong)-Viral Inactivation-AEX (strong)-Virus Inactivation: Nardini (13/519368) discloses a process for the production of IVIG starting from human plasma or an intermediate plasma fraction enriched in IgG comprising capturing IgG on a strong CEX followed by viral inactivation performed by solvent-detergent treatment, elution with a buffer having 0.25-1M NaCL followed by a strong AEX where elution is performed using an increased ionic strenght or by means of pH variation. 

CEX-AEX-Nano/Dialysis-3 virus inactivation steps: Starting with separation of cryo-precipitate, optinal absorption of blood coagulation factors and antithrombin, subject supernatant to modified Cohn-Oncley cold ethanol fractional. Following method 6 of Cohn, fibrinogen prcipitated in factionation Iand raw immunoglobulin spearated form raw albumin by a II & III preciption. In traditional ethanol fractionation, the next step would be separation of fraction III form IgG (decrases IgG yeild by about 25%. This step is omitted. The II &III paste dissolved and filtered. Precipate y-globulin by 25% ethanol at neutral pH similar to Chon II precipitation (IgG purity 75%, impurities are claases A and M), dissolve paste and filter to obtain filtrate which is treated for 1st virus inactivation step (S/D treatment), carboxy methyl cation exchange used to separate S/D reagents, elute under high pH (8.5), adjust ph to 6, apply to anion exchange (impurities like IgA bound), nanfilter flow through, diafiltrate against .25 mol/l glycine and concetrate. (Tschner, Vox Sanguinis (2007), 92,42-55.

Virus inactivation (by detergent/solvent) – CEX: Aghaie (Human Antibodies 19 (2010) pp. 1-6) discloses taking a fraction II paste, redesolving it in glycine buffer, virus inactivation using TNBP as a solvent and Tween 80 as a detergent and then using CEX on CM Sepharose FF. The elute was then dia/ultrafiltered after adding stabilizers. 

DF-Stabilize-Pasteurize-CEX-DF: Debart (US 13/838424) discloses a method for obtaining a liuqid IgG composition from an IgG solution which has previously been purified from human plasma by DF to reduce below concentration undersirable components such as ETOH, stabilizing the solution by the addition of sorbitol, pastuerisation by heating treating the solution at a temeprature of between 55-63C for 1-24 hours, selectively adsorbing high molecular weight aggregates and/or polymers by CEX such as a strong CEX resin and diafiltering the flow through. Optionlly the process can include one or more viral inactivation/elimination treatments to complement the heat treatment of the solution. 

Virus inactivation (S/D treatment) – CEX-AEX – Virus inactivation (pH): Teschner (Vox Sanguinis 2007, 92, 42-55 (2006) discloses an IGIV 10% manufacturing process starting with separation of the cryo-precipitate, followed by the optional adsorption of blood coagulation factors and antithrombin. The supernatant of these steps is then the starting material for a modified Cohn-Oncely cold thenaol fractional in that fibrinogen is precipitated in fraction I (following method 6 of Cohn) and raw immunoglobulin is separated from raw albumin by a II+III precipitation. In traditional ethanol fractional as carried out in Gammagard SD process, the next step would be the seapration of fraction III from IgG which is known to decrease the IgG yeild by about 25%. Thus this step is omitted and instead the II+III paste is dissolved in buffer anf filtered through a depth filter for depletion of lipids and undissovled proteins. From this filtrate, gama-globulin is precipitated by 25% ETOH at neutral pH similar to the Cohn II precipitation. The resulting intermediate has an IgG purity of aobut 75%. The main impurities are IgA and IgM. The paste is dissolved and filtered to obtain a clear filtrate for the first virus inactivation step, the S/D treatment (1% polysorbate 80, 3% Triton X 100 3% TnBP) and separated from IgG using carboxy-methyl CEX. Elution of IgG is conducted by moderately increasing the conductivity under alkaline conditions (higher pH 8.5). The diluted euate is adjusted to pH 6 and applied to an AEX (DEAE – diethylaminoethyl) in flow thorugh mode (IgA is bound while IgG flows through). The flow through is nanofiltered. The nanofiltrate is diafiltrated against glycine and concentration to 10% protein w/v. After sterile filtration the liquid is incubated for virus inactivation at pH 4.4-4.9 for 21-22 days.