WO2016130122A1

WO2016130122A1 - Prevention of hepatitis c virus recurrence using a human hepatits c immunoglobulin preparation

Info

Publication number: WO2016130122A1
Application number: PCT/US2015/015475
Authority: WO
Inventors: Shailesh CHAVAN; Chris Dougherty; Andrea Wartenberg-Demand; Frank SANANES; Eileen CHOI
Original assignee: Biotest Pharmaceuticals Corp
Current assignee: Biotest Pharmaceuticals Corp
Priority date: 2015-02-11
Filing date: 2015-02-11
Publication date: 2016-08-18
Anticipated expiration: 2017-08-11

Abstract

The present invention provides for a new therapeutic capable of treating HCV infections and of avoiding HCV recurrence in liver transplant patients, using a new human HCV- immunoglobulin preparation and applying a specific dosage regimen. This treatment schedule can potentially result in a cure for HCV patients such that HCV re-infection or recurrence of disease is gravely diminished or even absent.

Description

PREVENTION OF HEPATITIS C VIRUS RECURRENCE USING A HUMAN HEPATITIS C IMMUNOGLOBULIN PREPARATION

FIELD OF THE INVENTION

The present invention relates to the field of medicinal therapeutics and treatment of infectious diseases. In particular, the present invention relates to a new pharmaceutical composition for use in the treatment of hepatitis C virus (HCV) induced liver diseases and in the prevention of HCV recurrence after liver transplant.

BACKGROUND

Hepatitis C virus or HCV is an enveloped single strand RNA virus of the Flaviviridae family. It is the cause of hepatitis C and chronic liver disease including cirrhosis and liver cancer in humans. The virus replicates mainly in hepatocytes and peripheral blood mononuclear cells, leading respectively to destruction of the liver and immunological disorders in chronically infected HCV patients. The World Health Organization, or WHO, estimates that about 180 million people, or 3% of the world's population are chronically infected with HCV and two to four million people are newly infected each year.

HCV infection is responsible for 40-50% of all liver transplantations. However, in many of those subjects, HCV re-infection and disease recurrence occurs (cf. Charlton 2007, Current Gastroenterology Reports, Vol. 9(1 ):23-30). Antiviral treatment post liver transplantation may be associated with unwanted side effects for up to 6 months after transplantation and immune-suppression is generally needed to avoid rejection of the newly transplanted liver. There is currently no approved antiviral treatment post liver transplant and recurrence of disease through re-infection of the newly transplanted liver is universal in patients having residual HCV viral load at time of transplant. Recurrence of disease is furthermore associated with poor outcome for the subject.

Both monoclonal and polyclonal anti-HCV antibody (Ab) preparations or isolates have been tested for their efficacy in treating HCV infection or in preventing HCV re-infection after e.g. liver transplantation over the years. This research was reviewed in Mir et al., 2009 (Clin. Liver Dis. Vol. 13:477-486), Forde et al., 2009 (Clin. Liver Dis. Vol.13:391-401 ) and Ball et al., 2014 (Antiviral Res. Vol. 105(100): 100-1 1 1 ). In one of those studies by Davis and co- workers an open-label study with high doses of a human hepatitis C antibody enriched immune globulin product in patients undergoing liver transplantation for chronic hepatitis C (Davis et al., 2005, Liver Transplantation, Vol. 1 1 (8): 941-99) was described. The study was however not successful in that serum HCV RNA levels were not suppressed at either tested dose. Regarding monoclonal antibody preparations, we also would like to mention the publication of Schiano and co-workers of 2006 (Liver Transplantation Vol. 12:1381-9), reporting on the results of a Phase 2 randomized study with monoclonal antibody HCV- AbXTL68 in patients undergoing liver transplantation for HCV. A comparison is also made with a previously prepared human Hepatitis C Immune Globulin (hHCIG) isolate suggesting that the neutralizing activity in such pooled polyvalent immunoglobulin preparations is either too low or is blocked by other competing non-neutralizing antibodies to provide a protective response. Studies in Chimpanzees (cf. Krawczynski et al., 1996: J. Infectious Dis. Vol. 173:822-828) showed that HCIG preparations could delay infection with HCV, but not prevent it.

There is hence an unmet medical need for a method to treat HCV infection in liver transplant patients that prevents HCV re-infection after the liver transplantation.

SUMMARY OF THE INVENTION

The present invention provides for a new therapeutic capable of treating HCV infections and of avoiding HCV recurrence in liver transplant patients. The study of Davis et al., 2005 mentioned above, started from a patient population having high HCV viral blood titers and used an HCV-biased immunoglobulin preparation pooled from subjects with low HCV titers. An alternative administration scheme was used as well. The results were not promising at the time. The present inventors have now established that using a new hHCIG preparation and applying a new dosage regimen can potentially result in a cure for HCV patient, in that HCV re-infection or recurrence of disease is diminished or even absent.

In a first aspect, the invention provides a human hepatitis C immunoglobulin (hHCIG) preparation and a pharmaceutical composition comprising such a preparation, wherein said immunoglobulins are isolated from blood plasma obtained from subjects infected with HCV, specifically from subjects who are HCV antibody positive at the moment of donating plasma. The hHCIG preparation according to the invention hence comprises a pool of intravenous immunoglobulins (IVIGs) obtained from a pool of otherwise healthy donors who had an HCV infection and are HCV antibody positive. This directs the IVIGs towards multiple epitopes of HCV, creating a powerful HCV treatment tool.

Aspect 1. A purified human anti-hepatitis C immunoglobulin (hHCIG) preparation, isolated from pooled blood plasma substantially obtained from at least 200 different HCV antibody positive donors, or obtained from a group of donors comprising or consisting essentially of at least 200 different HCV-antibody positive donors, wherein the anti-HCV-antibody titer of each donor individual is at least 10 U/ml, preferably at least 15 U/ml, more preferably at least 20 U/ml, most preferably at least 25 U/ml and wherein the anti-HCV antibody titer of the pooled plasma is at least 400 U/ml, preferably at least 500 U/ml, or a pharmaceutical composition comprising such a hHCIG preparation.

Aspect 2. The hHCIG preparation or pharmaceutical composition according to aspect 1 , characterized in that the number of HCV antibody positive donors it at least 250, more preferably at least 300.

Aspect 3. The hHCIG preparation or pharmaceutical composition according to aspect 1 or 2, characterized in that the number of HCV antibody positive donors is not higher than 1000, particularly not more than 900, more particularly not more than 800, more particularly not more than 600 different donors.

Aspect 4. The hHCIG preparation or pharmaceutical composition according to anyone of aspects 1 to 3, characterized in that the HCV antibody positive donor group comprises both low and high HCV-antibody titer donors, preferably wherein the HCV-antibody titer of said high titer donors is at least 40 fold higher than the titer of the low titer donors, preferably at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least 100 fold higher.

Aspect 5. A pharmaceutical composition comprising the hHCIG preparation according to anyone of aspects 1 to 4, characterized in that it comprises a formulation of a 10% immunoglobulin solution, preferably comprising between 90 and 1 10 mg/ml immunoglobulin protein, preferably wherein more than 93%, more preferably more than 95% of said immunoglobulin protein is IgG, formulated in between 230 and 320 mM, preferably in about 300 mM glycine.

Aspect 6. The pharmaceutical composition according to aspect 5, characterized in that it is formulated at a pH of between 4 and 7, preferably of between 4.0 and 4.6, preferably of about 4.3.

Aspect 7. The hHCIG preparation or pharmaceutical composition according to anyone of aspects 1 to 6, characterized in that the donor plasma from which it is derived is HBV-DNA, HAV-RNA, and HIV-RNA non-reactive (negative) and Parvo B19 DNA levels do not exceed 1 x 10³ IU/ml.

Aspect 8. The hHCIG preparation or pharmaceutical composition according to anyone of aspects 1 to 7, characterized in that the donor plasma from which it is derived is (non- reactive) negative for Hepatitis B Surface Antigen (HBsAg), and HIV 1 , and HIV 2 antibodies.

Aspect 9. The hHCIG preparation or pharmaceutical composition according to any one of aspects 1 to 8, characterized in that it is HCV-RNA negative.

Aspect 10. The hHCIG preparation or hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 9, obtained from blood plasma of donors that have ALT- levels not higher than twice the upper level of the established normal range, preferably that have ALT-levels of below 219 U/ml, more preferably that have ALT levels below 150 U/ml. Aspect 1 1. hHCIG preparation or The hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 10, wherein said HCV virus is of anyone of genotypes 1 to 6, more preferably of genotypes 1 , 2, or 3.

Aspect 12. hHCIG preparation or The hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 1 1 , having an HCV antibody titer of more than 1000 U/ml, preferably of between 1000 and 4000 U/ml, preferably of between 1500 and 3000 U/ml.

Aspect 13. A container such as a bag, flask or a bottle suitable for infusion, e.g. comprising a sealed pierceable rubber stopper comprising the hHCIG preparation or pharmaceutical composition according to anyone of aspects 1 to 12. Aspect 14. A package or kit comprising a single or multiple container(s) according to aspect 12, and instructions for administration, preferably with instructions for administration according to the administration scheme of the present invention as defined herein.

Furthermore, the invention provides for a specific treatment scheme using an hHCIG composition as defined herein for treating HCV infection in a subject in need thereof.

Aspect 15. The hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12 for use in treating HCV infection in a subject.

Aspect 16. The hHCIG-comprising pharmaceutical composition for use according to aspect 15, wherein said subject is undergoing liver transplantation.

Aspect 17. The hHCIG-comprising pharmaceutical composition for use according to aspect 15 or 16, wherein said hHCIG-comprising pharmaceutical composition is administered in a dose of at least about 200 mg/kg, preferably of from 200 to 500 mg/kg, more preferably of from 300 to 400 mg/kg, or a dose equivalent thereto.

Aspect 18. The hHCIG-comprising pharmaceutical composition for use according to anyone of aspects 15 to 17, wherein said hHCIG-comprising pharmaceutical composition is administered in a dosage resulting in trough levels of hHCIG in subjects receiving treatment of at least 1 10 U of anti HCV antibodies per ml preferably of at least 120, 130, 140, 150, or 200 U of anti HCV antibodies per ml.

Aspect 19. A hHCIG-comprising pharmaceutical composition, preferably, the hHCIG- comprising pharmaceutical composition for use according to anyone of aspects 15 to 18, wherein said hHCIG-comprising composition is administered according to the following treatment regimen:

a) pre-treatment before the liver transplant surgery of the subject with anti-viral agents (AV) reducing the viral load in the subject to HCV-RNA levels of < 100 infective units/ml (lU/ml) prior to the liver transplantation,

b) at least 12, preferably 14 to 20 infusions of a hHCIG-comprising pharmaceutical composition, preferably the hHCIG-comprising therapeutic composition according to claim 1 to said subject, spread over a period of 6 to 15 weeks, comprising the following steps: b1 ) 2 to 4 infusions, preferably 3 infusions, surrounding the liver transplant surgery (day 1 )

b2) at least 9 infusions, preferably at least 12 infusions, spread over 6 to 15 weeks, preferably starting with daily intervals, preferably wherein the amount of each infusion is about 200 to 500 mg IgG per kg body weight;

or alternatively wherein said hHCIG-comprising composition is administered according to the following treatment regimen:

a) pre-treatment (i.e. before the liver transplant surgery) of the subject with anti-viral agents (AV) reducing the viral load in the subject to HCV-RNA levels of ≤ 100 infective units/ml (lU/ml), prior to the liver transplantation,

b) 16 infusions of the hHCIG-comprising therapeutic composition spread over a period of 10 weeks according to the following scheme:

b1 ) 3 infusions surrounding the liver transplant surgery (day 1 ), wherein the first dose is administered before liver transplant surgery, the second infusion is given during the anhepatic phase (when the diseased liver is removed), and the third infusion is given following 6 to 8 hours after said second infusion;

b2) followed by 5 infusions on consecutive days (e.g. on days 2, 3, 4, 5, and 6); b3) followed by 2 infusions twice per week (e.g. days 10, 14);

b4) followed by 4 infusions once per week (e.g. days 21 , 28, 35, and 42);

b5) followed by 2 infusions twice per month (e.g. days 56, and 70).

Aspect 20. The hHCIG-comprising pharmaceutical composition for use according to anyone of aspects 15 to 1 19, wherein the antiviral are virostatics targeting NS3/4a, NS5a and/or NS5b, preferably selected from the group comprising: Sofosbuvir, Telaprevir, Peg interferon, Ribavirin, Sofosbuvir, Simeprevir, or combinations thereof.

Aspect 21. The hHCIG-comprising pharmaceutical composition for use according to aspect 19 or 20, wherein said reduced viral load is obtained at least 4 weeks prior to the liver transplantation and maintained at said reduced viral load.

Aspect 22. The hHCIG-comprising pharmaceutical composition for use according to anyone of aspects 15 to 21 , wherein the antiviral treatment is initiated up to 6 months prior to the liver transplantation. Aspect 23. The hHCIG-comprising pharmaceutical composition for use according to anyone of aspects 15 to 22, wherein said reduced viral load is≤ 50 lU/ml, more preferably≤ 43 lU/ml prior to the liver transplantation.

Aspect 24. The hHCIG-comprising pharmaceutical composition for use according to anyone of aspects 10 to 18, wherein said patient is a subject infected with HCV, more specifically with HCV genotype 1 , 2, or 3, more specifically HCV genotype 1.

Aspect 25. The hHCIG-comprising pharmaceutical composition for use according to anyone of aspects 15 to 24, wherein said subject has chronic liver disease, liver cirrhosis, (strongly) decompensated liver cirrhosis, liver cancer, or hepatocellular carcinoma.

Aspect 26. The hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for use in neutralizing residual HCV in the body of a subject.

Aspect 27. The hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for use in treating HCV infection in a subject having had a needle-stick accident with an HCV contaminated needle.

Aspect 28. The hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for use in preventing HCV transfer from mother to embryo, foetus or baby, wherein the hHCIG-comprising pharmaceutical composition is administered to the mother.

Aspect 29. The hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for use in a co-treatment in poor responders to antiviral therapy, more specifically, in patients with strongly decompensated cirrhosis.

Aspect 30. The hHCIG-comprising pharmaceutical composition for use according to anyone of aspects 25 to 29, wherein said treatment is combined with other antiviral agents.

Aspect 31 : The invention also provides a method of treating HCV infection in a subject in need thereof comprising the administration of a dose of at least about 200 mg IgG /kg, preferably of from 200 to 500mg IgG /kg, more preferably of from 300 to 400 mg IgG /kg, or a dose equivalent thereto of the hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12 to said subject.

Aspect 32. The method according to aspect 31 , wherein the dose of said hHCIG-comprising pharmaceutical composition results in trough levels of hHCIG in subjects receiving treatment of at least 1 10 U of anti HCV antibodies per ml, preferably at least 120, 130, 140, 150, or 200 U of anti HCV antibodies per ml.

Aspect 33. The invention also provides for a method of treatment of HCV infection in a subject in need thereof comprising the administration of a dose of at least about 200 mg IgG /kg, preferably of from 200 to 500mg IgG /kg, more preferably of from 300 to 400 mg IgG /kg, or a dose equivalent thereto of a hHCIG-comprising pharmaceutical composition, wherein said hHCIG-comprising pharmaceutical composition is administered according to the following treatment regimen:

b) at least 12, preferably 14 to 20 infusions of a hHCIG-comprising pharmaceutical composition, preferably the hHCIG-comprising therapeutic composition according to claim 1 to said subject, spread over a period of 6 to 15 weeks, comprising the following steps:

b1 ) 2 to 4 infusions, preferably 3 infusions, surrounding the liver transplant surgery (day 1 )

or alternatively and preferably wherein said hHCIG-comprising composition is administered according to the following treatment regimen:

a) pre-treatment (i.e. before the liver transplant surgery) of the subject with anti-viral agents (AV) reducing the viral load in the subject to HCV-RNA levels of ≤ 100 infective units/ml (lU/ml), prior to the liver transplantation.

b1 ) 3 infusions surrounding the liver transplant surgery (day 1 ), wherein at least the first dose is administered before transplant because HCV resides predominantly in the old liver and can enter from the old organ into the circulation of the subject; b2) followed by 5 infusions on consecutive days (days 2, 3, 4, 5, and 6);

b3) followed by 2 infusions twice per week (e.g. days 10, 14);

b4) followed by 4 infusions once per week (e.g. days 21 , 28, 35, and 42);

b5) followed by 2 infusions twice per month (e.g. days 56, and 70), preferably wherein said hHCIG-comprising pharmaceutical composition is a composition according to anyone of aspects 1 to 12.

Aspect 34. The method according to aspect 31 or 32, wherein said hHCIG-comprising pharmaceutical composition is administered according to the following treatment regimen: a) pre-treatment before the liver transplant surgery of the subject with anti-viral agents (AV) reducing the viral load in the subject to HCV-RNA levels of ≤ 100 infective units/ml (lU/ml) prior to the liver transplantation,

b3) followed by 2 infusions twice per week (e.g. days 10, 14);

b4) followed by 4 infusions once per week (e.g. days 21 , 28, 35, and 42);

b5) followed by 2 infusions twice per month (e.g. days 56, and 70).

Aspect 35. The method of treatment according to anyone of aspects 31 to 34, wherein the antiviral are virostatics targeting NS3/4a, NS5a and/or NS5b, preferably selected from the group comprising: Sofosbuvir, Telaprevir, Peg interferon, Ribavirin, Sofosbuvir, Simeprevir, or combinations thereof.

Aspect 36. The method of treatment according to aspect 34 or 35, wherein said reduced viral load is obtained at least 4 weeks prior to the liver transplantation and maintained at said reduced viral load.

Aspect 37. The method of treatment according to any one of aspects 31 to 36, wherein the antiviral treatment is initiated up to 6 months prior to the liver transplantation.

Aspect 38. The method of treatment according to any one of aspects 34 to 37, wherein said reduced viral load is≤ 50 lU/ml, more preferably≤ 43 lU/ml prior to the liver transplantation.

Aspect 39. The method of treatment according to anyone of aspects 31 to 38, wherein said patient is a subject infected with HCV, more specifically with HCV genotype 1 , 2, or 3, more specifically HCV genotype 1.

Aspect 40. The method of treatment according to anyone of aspects 31 to 39, wherein said patient has chronic liver disease, liver cirrhosis, (strongly) decompensated liver cirrhosis, or liver cancer.

Aspect 41. A method for neutralizing residual HCV in the body of a subject comprising the administration of the hHCIG-comprising pharmaceutical composition according to any one of aspects 1 to 12.

Aspect 42. A method of treating a subject having had a needle-stick accident with an HCV contaminated needle, comprising the administration of the hHCIG-comprising pharmaceutical composition according to any one of aspects 1 to 12.

Aspect 43. A method for preventing HCV transfer from mother to embryo, foetus or baby comprising the administration of the hHCIG-comprising pharmaceutical composition according to any one of aspects 1 to 12 to the mother. Aspect 44. A method of preventing HCV-recurrence in re-transplant patients, comprising the administration of the hHCIG-comprising pharmaceutical composition according to any one of aspects 1 to 12 to the patient.

Aspect 45. A method of preventing HCV (re-)infection in a patient undergoing transplantation of a donor organ of unknown or positive HCV status, comprising the administration of the hHCIG-comprising pharmaceutical composition according to any one of aspects 1 to 12 to the patient.

Aspect 46. The method according to any one of claims 41 to 45, wherein said treatment is combined with other antiviral agents.

Aspect 47. The use of a hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12 in the manufacturing of a medicament for treating HCV infection in a subject.

Aspect 48. The use according to aspect 47, wherein said subject is undergoing liver transplantation.

Aspect 49. The use according to aspect 47 or 48, wherein said hHCIG-comprising pharmaceutical composition is administered in a dose of at least about 200 mg IgG /kg, preferably of from 200 to 500 mg IgG /kg, more preferably of from 300 to 400 mg IgG /kg, or a dose equivalent thereto.

Aspect 50. The use according to anyone of aspects 47 to 49, wherein said hHCIG- comprising pharmaceutical composition is administered in a dosage resulting in trough levels of hHCIG in subjects receiving treatment of at least 1 10 U of anti HCV antibodies per ml, preferably at least 120, 130, 140, 150, or 200 U of anti HCV antibodies per ml.

Aspect 51. The use according to anyone of aspects 47 to 50, wherein said hHCIG- comprising composition is administered according to the following treatment regimen:

a) pre-treatment before the liver transplant surgery of the subject with anti-viral agents (AV) reducing the viral load in the subject to HCV-RNA levels of ≤ 100 infective units/ml (lU/ml) prior to the liver transplantation,

b4) followed by 4 infusions once per week (e.g. days 21 , 28, 35, and 42);

b5) followed by 2 infusions twice per month (e.g. days 56, and 70).

Aspect 52. The use according to anyone of aspects 47 to 51 , wherein the antiviral are virostatics targeting NS3/4a, NS5a and/or NS5b, preferably selected from the group comprising: Sofosbuvir, Telaprevir, Peginterferon, Ribavirin, Sofosbuvir, Simeprevir, or combinations thereof.

Aspect 53. The use according to aspect 51 or 52, wherein said reduced viral load is obtained at least 4 weeks prior to the liver transplantation and maintained at said reduced viral load.

Aspect 54. The use according to anyone of aspects 47 to 53, wherein the antiviral treatment is initiated up to 6 months prior to the liver transplantation. Aspect 55. The use according to anyone of aspects 51 to 54, wherein said reduced viral load is≤ 50 lU/ml, more preferably≤ 43 lU/ml prior to the liver transplantation.

Aspect 56. The use according to anyone of aspects 47 to 55, wherein said patient is a subject infected with HCV, more specifically with HCV genotype 1 , 2, or 3, more specifically HCV genotype 1 .

Aspect 57. The use according to anyone of aspects 47 to 56, wherein said subject has chronic liver disease, liver cirrhosis, (strongly) decompensated liver cirrhosis, liver cancer, or hepatocellular carcinoma.

Aspect 58. Use of the hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for the manufacturing of a medicament for neutralizing residual HCV in the body of a subject.

Aspect 59. Use of the hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for the manufacturing of a medicament for treating HCV infection in a subject having had a needle-stick accident with an HCV contaminated needle.

Aspect 60. Use of the hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for the manufacturing of a medicament for preventing HCV transfer from mother to embryo, foetus or baby, wherein the hHCIG-comprising pharmaceutical composition is administered to the mother.

Aspect 61 . Use of the hHCIG-comprising pharmaceutical composition according to anyone of aspects 1 to 12, for the manufacturing of a medicament for in a co-treatment in poor responders to antiviral therapy, more specifically, in patients with strongly decompensated cirrhosis.

Aspect 62. The use according to anyone of aspects 58 to 61 , wherein said treatment is combined with other antiviral agents.

Aspect 63. Preparation of an hHCIG preparation or composition with a process comprising the following steps:

a) thawing of frozen plasma comprising anti-HCV antibodies, preferably at 20-40 °C, b) reduction of insoluble aggregates from the thawed plasma,

c) treatment of the plasma depleted of insoluble aggregates with solvent/detergent, d) isolating, preferably by ethanol fractionation, from the solvent/detergent treated plasma an IgG containing solution comprising at least 93%, preferably at least 95%, most preferably at least 98% pure IgG based on the total protein content,

e) optionally, further clarification of the IgG containing solution,

f) optionally, ultrafiltration and diafiltration of the IgG containing solution,

g) treatment with a solvent/detergent solution,

h) reduction of the solvent/detergent from the solution of step g),

i) optionally further purification of the IgG-containing solution by chromatography to reduce, residual plasma-derived impurities such as PKA, IgA, and IgM,

k) nanofiltration of the IgG solution,

I) ultrafiltration and diafiltration to the IgG-containing solution,

m) optionally formulating said hHCIG preparation into a pharmaceutical composition, and n) optionally the pharmaceutical composition is filled into a suitable container

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Schematic representation of a preferred treatment scheme of the hHCIG preparation according to the invention. The scheme generally represents three phases, a pre-transplant phase (pre-LT), a transplant phase (LT), and a post-transplant phase (post- LT).

Figure 2: Schematic representation of the IgG-population present in the hHCIG preparation according to the invention. In the left hand panel, a typical situation is depicted where a subject is receiving anti-HCV IVIGs from a single donor. The viral epitopes recognized by the IgGs will be limited and a number of HCV variants or mutants will not be affected by the treatment. In the right hand panel, due to the use of a pool of donors, the IgGs will typically recognize many more HCV epitopes and hence will be much more effective in avoiding re-infection of the subject.

Figure 3: Schematic overview of patient results of the control group. Light grey blocks indicate virus-free periods, dark grey blocks indicate viral re-infection. Figure 4: Schematic overview of patient results of the 200mg/kg group. Light grey blocks indicate virus-free periods, dark grey blocks indicate viral re-infection.

Figure 5: Schematic overview of patient results of the 300mg/kg group. Light grey blocks indicate virus-free periods, dark grey blocks indicate viral re-infection.

Figure 6: Flow-chart representing the main steps of the method of producing the hHCIG preparation as defined herein.

Figure 7: Studies on minimal effective concentration of hHCIG. Figure 7a shows the Median HCV Antibody Titer in Control Subjects Reinfected (Positive) and Viral Free (Negative); Figure 7b shows Individual (single dots) and Median (line) Baseline HCV Ab Levels Per Treatment and Reinfection (R); Figure 7c shows HCV Antibody and RNA Over Time by Subject (200 mg/kg, Reinfected) in Patient 25; Figure 7d shows HCV Antibody and RNA Over Time by Subject (200 mg/kg, Reinfected) in Patient 33; Figure 7e shows HCV Antibody and RNA Over Time by Subject (200 mg/kg, Reinfected) in Patient 4; Figure 7f shows HCV Antibody and RNA Over Time by Subject (200 mg/kg, Reinfected) Patient 15.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Skilled Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than the one understood by a person of ordinary skill in the art.

Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks, to the general background art referred to above and to the further references cited therein.

As used herein, the singular forms 'a', 'an', and 'the' include both singular and plural referents unless the context clearly dictates otherwise.

The terms 'comprising', 'comprises' and 'comprised of as used herein are synonymous with 'including', 'includes' or 'containing', 'contains', and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term 'about' as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +1-5% or less, more preferably or less, and still more preferably +/-

0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier 'about' refers is itself also specifically, and preferably, disclosed.

The term "HCV" is synonymous to Hepatitis C virus and encompasses all genotypes. More specifically the term comprises the following genotypes: Genotype 1 (including 1 a, 1 b and 1 c), Genotype 2 (including 2a, 2b, 2c and 2d, Genotype 3 (including 3a, 3b, 3c, 3d, 3e and 3f), Genotype 4 (including 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i and 4j), Genotype 5 (5a), Genotype 6 (6a), as well as further mutants or variants thereof called quasispecies. For more information we refer to Simmonds et al., 1993 (Journal of General Virology, 74, 2391 - 2399) reporting on a consensus HCV classification scheme. HCV genotypes 2 and/or 3 are usually more responsive to standard anti-viral therapies in contrast to HCV genotype 1 , which is the most abundant HCV genotype.

The HCV-genotype to be treated is not to be seen as being limiting since the hHCIG will show a certain amount of cross-reactivity with all genotypes due to the pooling of donor plasma. In Europe and North and South America the majority of HCV infected subjects have genotypes 1 , 2, and 3, so in preferred embodiments HCV genotypes 1 , 2, and/or 3 are treated. Genotype 4 is found typically in the Middle East and North and Central Africa. Genotype 6 is common in South-east Asia, while Genotype 5 is responsible for at least 30% of cases in South Africa. The efficacy of the hHCIG preparation to treat diverse HCV genotypes may be further improved by including blood plasma donors with said other HCV genotypes into the pool of donors from which the hHCIG preparation is obtained.

The term "HCV- related disease" encompasses all HCV-induced or related diseases and disorders. Some examples of HCV-related diseases and disorders are given herein: chronic hepatitis C, liver cirrhosis, liver injury, liver disease, end-stage liver disease, liver cancer, hepatocellular carcinoma, B-cell non-Hodgkin's lymphoma, cryoglobulinemia, lichen planus and porphyria cutanea tarda. Liver cirrhosis occurs when normal liver cells are replaced by scar tissue as a result of chronic liver disease due to chronic HCV infection. Chronic hepatitis C is a long-lasting liver infection caused by the hepatitis C virus. During the first 6 months of the infection, the disease is called acute hepatitis. Most people with acute hepatitis C (80%) become chronic hepatitis C patients. Hepatitis C virus (HCV) infection is also associated with the development of hepatocellular carcinoma, the most common type of liver cancer. Another associated condition is cryoglobulinemia, due to the presence of abnormal antibodies (called cryoglobulins) due to hepatitis C virus stimulation of lymphocytes, which form deposits in small blood vessels causing inflammation (vasculitis) in tissues throughout the body including the skin, joints, and kidneys (glomerulonephritis). Also B-cell non-Hodgkin's lymphoma, a cancer of the lymph tissue, has been associated with chronic hepatitis C virus. The cause is thought to be the excessive stimulation by the hepatitis C virus of B-lymphocytes, which results in the abnormal reproduction of lymphocytes. Also two skin conditions, lichen planus and porphyria cutanea tarda, have been associated with chronic hepatitis C virus. In addition, up to 65% of hepatitis C virus patients have autoimmune antibodies, such as anti-nuclear antibodies, anti-smooth muscle antibodies, and rheumatoid factors. Chronic hepatitis C is sometimes also associated with type 2 diabetes and insulin resistance. The patients that can be treated with the hHCIG treatment scheme according to the invention can be all subjects being infected with HCV, more specifically with HCV genotype 1 , 2, or 3, more specifically HCV genotype 1.

Patients can or cannot have beginning or progressed liver disease, the stage (e.g. defined by the MELD score or Child-Pugh-Score) or progression of the liver disease is not critical for patient selection, since typically patients in need of a liver transplant will be treated. Patients with (strongly) decompensated cirrhosis can also be successfully treated with the hHCIG treatment scheme according to the invention. The MELD (Model for End-stage Liver Disease) Score is a scoring system for assessing the severity of chronic liver disease and is used in prognosis and in prioritizing liver transplant patients. The formula is (0.957 ^* ln(Serum Creatinine) + 0.378 ^* ln(Serum Bilirubin) + 1.120 ^* ln(international normalized ratio for prothrombin time - INR ) + 0.643 ) ^* 10. In case of recent hemodialysis, the value for Creatinine is automatically set to 4.0. If any score is <1 , the MELD assumes the score is equal to 1 to prevent negative results. In interpreting the MELD Score in hospitalized patients, the 3 month mortality is (Wiesner et al., 2003, Gastroenterology, vol. 124 (1 ) pp. 91-6):

• 40 or more— 71.3% mortality

• 30-39— 52.6% mortality

• 20-29— 19.6% mortality

• 10-19— 6.0% mortality

• <9— 1.9% mortality

Alternative to the MELD score, the Child-Pugh-Score can be used as a criterion for the severity of liver disease, especially liver cirrhosis. It is based on a combination of scores of total bilirubin, serum albumin, prothrombin time (PT), ascites, and hepatic encephalopathy. Chronic liver disease is classified into Child-Pugh class A through C, employing the added score from above (cf. Child CG, Turcotte JG (1964), The liver and portal hypertension. Philadelphia: Saunders, pp. 50-64; Pugh RN et al., 1973, The British journal of surgery 60 (8): 646-9).

Class One year survival Two year survival

A 100% 85%

B 81 % 57%

C 45% 35%

Liver transplantation implies the surgical procedure replacing the injured liver of the subject under treatment with a matching donor liver, or a part of a donor liver (e.g. in case of living donor transplantation). Liver transplantation is currently performed routinely as a treatment of last resort for patients with end-stage liver disease. Liver transplantation may be performed with liver donation after brain or cardiac death or with a liver segment donation from a living donor. Patients are prioritized for transplant by mortality risk and severity of illness criteria. The severity of illness is determined by the model for end-stage liver disease (MELD) and/or Child-Pugh scores. Liver transplant surgery is generally followed by immunosuppression treatment to avoid donor liver (allograft) rejection. Said treatment is well known in the medical world and is rather uniform form all solid organ transplantations. Examples of such immunosuppressive post liver transplantation treatments can be corticosteroids, calcineurin inhibitors such as tacrolimus or cyclosporine, purine antagonists such as mycophenolate mofetil, mTOR inhibitors such as everolimus or sirolimus, or combinations thereof.

"Recurrence" or "re-infection" as used herein are used interchangeably. According to the present invention, a patient is considered to have recurrence of HCV or a re-infection, if the plasma titer of HCV RNA reaches more than 100 lU/ml within 24 weeks of the transplantation, particularly within 12 weeks of the transplantation. Patients having a titer of less than 100 U/ml at 12 weeks after transplantation are highly likely to have a titer of less than 100 lU/ml also at 24 weeks of after transplantation. A patient having a titer of less than 100 lU/ml at 24 weeks after transplantation is considered to be cured from HCV.

The blood plasma donors for producing the hHCIG preparation according to the invention are otherwise healthy individuals that are currently or were previously infected with HCV and are HCV antibody positive.

In contrast to typical hyperimmune globulins, the donors can be HCV positive, meaning that the donors as well as the individual donations may contain active virus. There is no particular limit on the amount of viral RNA in the donor.

Preferably said plasma donors are donors that have an HCV antibody titer level of at least 10 U/ml, preferably at least 15 U/ml, more preferably at least 20 U/ml, most preferably at least 25 U/ml, as measured for example by an ELISA which uses peptides from the HCV virus as bait proteins in order to bind anti-HCV antibodies such as the Vitros ECi method. Said antibody titer can be directly measured in plasma samples of the donors. Said Vitros ECi assay can use a reference standard which can be determined by measuring the titers in blood plasma of a suitable random sample of HCV-RNA positive subjects, particularly at least 50 subjects, more particularly at least 80, more particularly at least 150 subjects. Next, the anti-HCV antibody titers of the subjects in said sample are measured. The titers of the 30% subjects with the lowest titer and the titers of 30% of subjects with the highest titer are then excluded from further analysis, so that a subgroup of titers is formed. Said average titer is then calculated as the arythmetic mean of the titers of said subgroup. Said average titer obtained by this method is defined as 500 U/ml of blood plasma.

Preferably, said subjects in the sample are HCV genotype 1 positive. Preferably, said subjects are from 20 to 35 years old. The term "otherwise healthy" means the subjects should not suffer from other morbidities, particular not from other morbidities affecting the immune system. Particularly said subjects should comply with the criteria specified for plasma donors according to the present invention. More particularly, said subjects should not have an ALT exceeding twice the upper normal level, particularly not exceeding 219 U/ml, as described elsewhere in this specification. In the present context, the term "not antiviral treated" HCV-RNA positive subjects means that the said subjects have not been treated with an antiviral agent active against HCV, particularly with an antiviral agent as defined in the present specification.

HCV RNA positive subjects can be identified by any suitable assay, such as the National Genetics Institute UltraQualt-1000 method.

In addition the values may be compared or normalized to values found on a population level. If one looks to donors according to the donor acceptance criteria (ALT not exceeding twice the upper normal level, particularly not exceeding 219 U/ml, HBV-DNA, HAV-RNA, ParvoB19-DNA and HIV-RNA negative as described in this specification) and who have not been treated yet with antiviral agents, most of such donors have a titer of between of between 300 and 700 U/ml. The highest titers encountered in some donors according to the donor acceptance criteria who have not been treated yet with antiviral agents are in a range of 900 to 2000 U/ml, in exceptional cases approximately 4000 U/ml.

In the course of the invention, it was found that the HCV antibody titers of individual donors are fairly stable over time and that not every donation of one donor has to be titered. It is sufficient to measure the titer every 2nd, 3rd, or 4th donation, or every two to three weeks. Missing values can be considered to be equal to the last measurement. This spacing of measurements still allows to achieve the calculated target titer in the plasma pool. Furthermore the blood plasma donors should be free from active liver disease, e.g. free of signs and symptoms of liver disease. Donors preferably do not have Alanine Aminotransferase (ALT) levels higher than twice the upper level of the established normal range. The normal range is typically between 0 and 75 U/ml. The normal range can be established by each laboratory individually according to procedures known in the art. However, a donor should preferably not have an ALT level of above 219 U/ml, independent of individual range of the laboratory. The alanine aminotransferase (ALT) level in a blood plasma sample is indicative of the disease state of the patient. ALT is also known as serum glutamic pyruvic transaminase (SGPT) and is an enzyme which catalyzes the reversible transfer of an amino group from alanine to a-ketoglutarate, generating pyruvate and glutamate. ALT is concentrated in the liver, more precisely in the hepatocytes. Typically, HCV infections will destroy the hepatocytes, which subsequently release ALT in the elevated blood, making the presence of the latter in blood an important indication for liver injury or active liver disease. The enzyme activity of ALT can be measured in plasma using known methodologies such as quantitative enzyme-linked immunosorbent assays (ELISA).

Said plasma donors and their corresponding plasma units are tested for HBV-DNA, HAV- RNA, ParvoB19-DNA and HIV-RNA by current state of the art test kits such as for example the ROCHE cobas® TaqScreen MPX, a qualitative in vitro test for the direct detection of Human Immunodeficiency Virus Type 1 (HIV-1 ) Group M RNA, HIV-1 Group O RNA, Human Immunodeficiency Virus Type 2 (HIV-2) RNA and Hepatitis B Virus (HBV) DNA in human plasma, and the ROCHE cobas TaqScreen DPX, an in vitro nucleic acid amplification test for the direct quantitation of human parvovirus B19 DNA and the direct qualitative detection of Hepatitis A virus (HAV) RNA in human plasma. For HIV, HBV & HAV acceptable results are those units found non-reactive (negative) based on the assay's established cut-off and Limit of Detection (LOD). In most assays, the LOD is below 15 RNA copies/ml or lower such as less than 10 RNA copies/ml or less than 5 RNA copies/ml. In the case of Parvo B19, the viral load must not exceed 1 x 10³ lU/ml.

The donors and corresponding plasma units should also have been found to be negative for Hepatitis B Surface Antigen (HBsAg), and for the HIV 1 , and HIV 2 antibodies by commercially available qualitative test kits such as for example Abbot PRISM HBsAg assay or the Abbott PRISM HIV O Plus assay. In this case, negative is defined as those specimens with net counts less than the cutoff value established by the criteria of the Abbott PRISM assay. Most donors according to the above criteria and who have not been treated yet with antiviral agents have a titer of between of between 300 and 700 U/ml. The highest titers encountered in some donors according to the above criteria and who have not been treated yet with antiviral agents are in a range of 900 to 2000 U/ml, in exceptional cases approximately 4000 U/ml.

In order to obtain a preparation comprising as many different anti-HCV antibodies as possible, plasma of a number of donors is pooled. As used herein, the pool of donors from which the blood plasma is obtained to produce the hHCIG preparation is not to be seen as being limited to a specific number of donors. In said pool of donors, individual donors can have highly varying antibody titers, i.e. ranging from 25 U/ml to up to several thousand U/ml. Such diversity may be advantageous as it may reflect different immune responses and thus increase diversity of antibodies in the pool. Yet, the goal of the present invention is to reach a hHCIG preparation or pharmaceutical composition that comprises a sufficiently high anti- HCV antibody titer, of preferably above 400 U/ml, more preferably above 500 U/ml, such as above 1000 U/ml, preferably of between 1000 and 3000 U/ml (all preferably based on a 10% w/v total IgG content in the preparation) as measured by the Ortho's Vitros ECi methodology. The antibody titer in plasma can be measured using established test kits, such as the Abbott HCV-ELISA or the Ortho's Vitros ECi methodology. Said composition should comprise IgG derived from at least 200, preferably at least 250, more preferably at least 300 different HCV-antibody positive donors.

In general, such test methods use recombinant peptides encoded by the Hepatitis C Virus. A two-stage immunometric technique is used. In the first stage, HCV antibody present in the sample is allowed to bind with the recombinant HCV antigens coated on a solid support. Unbound sample is removed by a wash step. In a second stage, a labeled antibody conjugate is added which binds to any human antibodies. Any unbound conjugate is washed off and the amount of label present on the solid support is measured, which is directly proportional to the concentration of the element in a testing solution.

Any quantitatively detectable label can be used in conjugation with the antibody. The label can e.g. be (auto fluorescent or can be an molecule or enzyme that reacts with a substrate. One well know example is horseradish peroxidase (HRP), which is used in combination with a luminogenic substrate such as a combination of a luminol derivative, peracid salt and an electron transfer agent. The HRP in the antibody conjugate catalyzes the oxidation of the luminol derivative resulting in a light signal. The electron transfer agent increases the level of light produced and prolongs its emission. The light produced is measured in adjusted light units (ALUs). The amount of light generated is directly proportional to the concentration of the element in a testing solution.

Anti-HCV antibodies are defined as being the group of antibodies that specifically binds HCV antigens. Such antigens are derived from HCV structural proteins which form the viral particle which include the core protein and the envelope glycoproteins E1 and E2, or the HCV nonstructural proteins including the p7 viroporin, the NS2 protease, the NS3-4A complex, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. The antibody titer of the hHCIG preparation according to the present invention generally encompasses the IgG antibodies specific for HCV epitopes. Such HCV-specific IgG antibodies can comprise both neutralizing and non-neutralizing antibodies. Neutralizing antibodies typically are directed towards the envelope glycoproteins E1 and E2 and can inhibit or neutralize the infection of the virus.

The pooled plasma starting material, from which the hHCIG preparation is made, preferably has an anti-HCV antibody titer (called the Pool Calculated Titer) of at least 400 U/ml, such as 450 U/ml, more preferably at least 500 U/ml, preferably between 400 and 1000, such as between 400 and 800 U/ml, between 400 and 700, or between 400 and 600 U/ml. This value can be calculated from the titer and volume of the different donation- units by using the following formula: Pool Calculated Titer = (Unit Volume^*Unit Ttiter) / Total Pool Volume. The total pool volume is not particularly limited in size, e.g. it may range from 800 to up to 4500 liters.

The term "pooled plasma" as defined above does not mean that the pool is actually manufactured as a single pool. It may be advantageous to manufacture the pool in subpools, e.g. the upfront S/D treatment as described elsewhere may be performed on subpools. The pooled plasma thus can be blended from subpools or at later stages of manufacturing the hHCIG preparation. Indeed, also the hHCIG preparation or the final composition may be blended from different hHCIG preparations or compositions.

Preferably, the pool contains plasma from at least 200, at least 250, or at least 300 donors. Although previous studies have suggested that more than 1000 donors would be needed in order to obtain an effective HCIG preparation, the present invention shows that a cut-off of at least about 300 donors can be sufficient. In general, a higher number of different donors is preferred in order to increase the potential diversity of virus epitopes recognized by the preparation. The inventors have found that a pool from about 300 donors contains sufficient antibody diversity for efficient treatment according to the treatment regimen of the invention. The number of donors can hence vary between 200 to 1000, preferably between 250 and 900, or between 300 and 800 donors, more preferably between 300 and 600 donors. The number of donors and the difference in donor HCV antibody titer results in a pool of antibodies that have a great diversity towards HCV.

The plasma pool can be HCV-positive, since it will undergo several purification steps which will remove the viral RNA from the final preparation. The inclusion of both low anti-HCV antibody titer and high anti-HCV antibody titer donor plasma, leads to a final plasma pool with a Pool Calculated Titer greater than or equal to 400 or greater than or equal to 500 U/ml anti-HCV antibodies. This ensures high HCV-epitope variability (the number of donors with different backgrounds is high), while maintaining a high binding capacity of the pooled plasma.

Said pooled plasma should be HBV-DNA, HAV-RNA, ParvoB19-DNA and HIV-RNA negative. Negative in this respect means non-detectable, i.in particular non-detectable according to FDA (Federal Food and Drug Administration) guidelines, e.g. less than 10⁴ U/ml for ParvoB19-DNA and less than 15, preferably less than 10, more preferably less than 5 RNA-copies/ml. for the other viruses. The pooled plasma must also be negative for Hepatitis B Surface Antigen (HBsAg) and HIV 1 , and HIV 2 antibodies. Negative in this respect means non-detectable, i.e. in particular non-detectable according to FDA (Federal Food and Drug Administration) guidelines, i.e. less than 15 U/ml.

In the context of the invention it was also found that for an effective composition, it is not necessary to obtain plasma only from donors having a particularly high titer.

Therefore, the present invention also relates to an hHCIG-comprising pharmaceutical composition, wherein the composition has an anti-HCV antibody titer (based on mg of total IgG), which (numerical value) corresponds to at least 0.03-fold the (numerical) value of the titer of the average anti-HCV antibody titer measured in blood plasma of otherwise healthy not antiviral treated HCV-RNA positive subjects (based on ml of undiluted plasma), and wherein the composition comprises anti-HCV antibodies derived from at least 200, more preferably at least 250, most preferably at least 300 different anti-HCV antibody positive donors. Preferably, at least 10% of said different donors have an anti-HCV antibody titer measured in blood plasma of at least 1.8-fold of the average anti-HCV antibody titer measured in blood plasma of otherwise healthy not antiviral treated HCV-RNA positive subjects.

Preferably, at least 10% of said different donors have an anti-HCV antibody titer measured in blood plasma of at less than 0.4-fold of the average anti-HCV antibody titer measured in blood plasma of otherwise healthy not antiviral treated HCV-RNA positive subjects

Preferably said antibody titer of the composition is at least 0.035-fold, more preferably at least 0.036-fold the titer of the average anti-HCV antibody titer measured in the plasma of said subjects.

Preferably said antibody titer of the composition is 0.035-fold to 0.060-fold or 0.035-fold to 0.050-fold, more preferably 0.036-fold to 0.060-fold or 0.036-fold to 0.050-fold the titer of the average anti-HCV antibody titer measured in the plasma of said subjects.

In this context, said titers can be measured using any immunoassay, such as a quantitative ELISA. Particularly, said assay should be based on the three recombinant hepatitis C virus encoded antigens c22-3, c200 and NS5, such as used in Ortho's Vitros ECi method.

Said "average titer" in the present context can be determined by measuring the titers of a suitable random sample of plasma from HCV-RNA positive subjects, particularly at least 50 subjects, more particularly at least 80, more particularly at least 150 subjects. Next, the anti- HCV antibody titers of the subjects in said sample are measured. The titers of the 30% subjects with the lowest titer and the titers of 30% of subjects with the highest titer are then excluded from further analysis, so that a subgroup of titers is formed. Said average titer is then calculated as the arythmetic mean of the titers of said subgroup. Said average titer may also serve as a 500 U/ml reference standard for the purposes of the present invention

Preferably, said subjects in in the sample are HCV genotype 1 positive. Preferably, said subjects are from 20 to 35 years old. The term "otherwise healthy" means the subjects should not suffer from other morbidities, particular not from other morbidities affecting the immune system. Particularly said subjects should comply with the criteria specified for plasma donors according to the present invention. More particularly, said subjects should not have an ALT exceeding twice the upper normal level, particularly not exceeding 219 U/ml, as described elsewhere in this specification. In the present context, the term "not antiviral treated" HCV-RNA positive subjects means that the said subjects have not been treated with an antiviral agent active against HCV, particularly with an antiviral agent as defined in the present specification.

HCV RNA positive subjects can be identified by any suitable assay, such as the National Genetics Institute UltraQualt-1000 method

In a further aspect the invention provides for a method of producing a hHCIG preparation as defined herein. In essence, the method comprises the steps of collecting or obtaining blood plasma from high HCV titer blood donors ("hot" plasma), an initial viral inactivation (resulting in a "cold" plasma) and purifying the immunoglobulins from said plasma pool using an IVIG purification process such as cold alcohol fractionation processes known in the art. Such processes use three subsequent cold ethanol precipitation steps, resulting in a fraction (such as a "Fraction III" supernatant, which comprises purified IgG antibodies from the plasma. It was found that the upfront solvent/detergent treatment is not detrimental to the subsequent cold ethanol precipitation steps. This precipitation step is followed by depth filtration, viral inactivation, column chromatography, and filtration steps resulting in the final hHCIG preparation of the invention. The final preparation can be concentrated to a desired concentration. Preferably, a concentration of 4-12%, most preferably about 10% IgG will be present in the final preparation.

The hHCIG preparation as defined herein can be prepared with a process comprising the following steps:

a) Thawing of frozen plasma comprising anti-HCV antibodies, preferably at 20-40 °C b) reduction of insoluble aggregates from the thawed plasma

c) treatment of the plasma depleted of insoluble aggregates with solvent/detergent d) isolating, preferably by ethanol fractionation, from the solvent/detergent treated plasma an IgG containing solution comprising at least 93%, preferably at least 95%, most preferably at least 98% pure IgG based on the total protein content.

e) optionally, further clarification of the IgG containing solution

f) optionally, ultrafiltration and diafiltration of the IgG containing solution

g) treatment with a solvent/detergent solution

h) reduction of the solvent/detergent from the solution of step g) i) optionally further purification of the IgG-containing solution, preferably by chromatography, particularly to reduce at least one residual plasma-derived impurity, particularly chosen from the group consisting of as PKA, IgA, and IgM

k) nanofiltration of the IgG solution,

I) ultrafiltration and diafiltration of the IgG-containing solution

m) optionally formulating said hHCIG preparation into a pharmaceutical composition n) optionally the pharmaceutical composition is filled into a suitable container

The above process may comprise additional steps which are in the routine of the skilled person, chromatography steps and or filtration or centrifugation steps. The conditions of each step such as particular pH values or salt concentrations can be determined by the skilled person. Further guidance and preferred conditions are outlined in this specification, in particular below and in the Examples.

Preferably, the process contains no step of separating a cryoprecipitate from the plasma. The thawing of the plasma can be carried out by heating the frozen plasma by air-flow, in a water bath, or using any other suitable heating means. The plasma of different donations may be pooled after or during the thawing.

The reduction of insoluble aggregates can be done by filtration (e.g. depth filtration), centrifugation or other suitable techniques. Reduction of insoluble aggregates is beneficial to increase effectiveness of the subsequent solvent detergent treatment. In essence, the plasma can contain insoluble aggregates (e.g. clots such as fibrin clots), which may shield viruses contained within the aggregates from the solvent/detergend (S/D) treatment. The plasma of different donations may be pooled before or after the filtration.

The step of solvent/detergent treatment is intended to inactivate the HCV virus present in any HCV virus positive plasma. This step is important as the plasma for the preparation according to the invention is allowed to be derived from HCV positive donors. Solvent/detergent treatment of plasma as such is generally known in the art. In particular, treatment can be done with any suitable solvent/detergent such as Tri-n-butyl phosphate (TNBP) and Polysorbate (such as Tween-80) or polyethylene oxide (such as Triton X-100). More preferably the treatment is with a solvent/detergent solution comprising 0.3% to 1.3% w/w, preferably 0.9% to 1.3% w/w, more preferably about 1.1 % w/w Tri-n-butyl phosphate and 0.9 - 1.3% w/w, preferably about 1.1 %, Triton X-100. The treatment is meant to disrupt the virus envelope of potential HCV virus and to allow RNAse enzymes to digest the HCV RNA. This will essentially inactivate the virus. RNAses are naturally present in the plasma pool, but they could also be added. This step should have a certain duration and temperature to be effective, i.e. ensuring the viral coat is opened and RNAses are allowed to digest the viral RNA. Preferably, treatment is carried out by incubation at 25-35 °C, more preferably at 28-32 °C. Preferably said incubation is for at least 2 hours, more preferably for at least 4 hours, more preferably at least 6 hours, more preferably about 8 hours, all at a temperature of between 28-32 °C, more preferably at about 30 °C. The appropriate time and temperature can be determined by the skilled person monitoring the amount of residual virus RNA. Preferably, the solvent/detergent treatment is carried out in a manner that the amount of residual virus RNA is equal or less than 500 copies, more preferably equal or less than 100 copies/ml. The plasma of different donations may be pooled before or after the S/D treatment.

Alternatively to this solvent/detergent treatment of the thawed plasma, already the fresh plasma may be treated with solvent/detergent.

The IgG containing solution can be obtained by any suitable method known for purification of IgG from plasma preparations, such as precipitation, chromatography or other methods as long as they do not substantially denature or affect the ability of the anti-HCV antibodies comprised to bind to HCV virus proteins. A particularly economical method suitable for scale is ethanol fractionation. In particular, the solution can obtained by cold ethanol fractionation, which is well known in the art. Such fractionation can be Cohn/Oncley fractionation or Kistler/Nitschmann fractionation. In cold ethanol fractionation, a series of ethanol based precipitation and separation steps are performed (for example: Cohn Fraction I, Cohn Fraction ll/lll, Cohn Fraction ll/lllw) until the IgG containing fraction is isolated which comprises 93%, preferably at least 95%, most preferably at least 98% pure IgG based on the total protein content (e.g. a Cohn Fraction III supernatant),

If necessary, the IgG containing solution may be further clarified by a suitable method, e.g. depth filtration.

Optionally, a step of ultrafiltration/diafiltration of the IgG containing solution, if applicable of the clarified IgG containing solution, is performed. This step is can serve to reduce the amount of residual ethanol from an earlier ethanol fractionation step. Such steps are generally known to person skilled in the art.

The IgG containing solution is further subjected to a (second) solvent/detergent treatment. Solvent/detergent treatment of IgG solutions, such as IgG solutions obtained by cold ethanol fractionation, is generally known in the art. The treatment is meant to disrupt the virus envelope of residual enveloped virus in the IgG containing solution. This S/D treatment step serves to essentially inactivate residual enveloped virus. Suitable conditions are known to the skilled person and can In particular, treatment can be done with any suitable solvent/detergent such as Tri-n-butyl phosphate (TNBP) and Polysorbate (such as Tween- 80) or polyethylene oxide (such as Triton X-100). More preferably the treatment is with a solvent/detergent solution comprising 0.2% to 0.4% w/w, preferably about 0.3% w/w Tri-n- butyl phosphate and 0.8-1.2% w/w, preferably about 1.0%, Triton X-100. Preferably, treatment is carried out by incubation at 25-35 °C, more preferably at 26-30 °C. Preferably said incubation is for at least 2 hours, more preferably for at least 4 hours, more preferably at least 6 hours, more preferably at least 8 hours, all at a temperature of between 26-30°C, more preferably at about 28°C. There is no particular limit to the duration of S/D treatment, up to 72 hours have been shown to be acceptable.

Removal of the solvent/detergent can be done by methods known in the art, for example reverse phase column chromatography (such as C18 chromatography), cation exchange, or oil extraction).

The optional further purification step of the IgG-containing solution can be used to reduce residual plasma-derived impurities such as PKA, IgA, and IgM. The step can be carried out by chromatography such as anion exchange and/or Q-Sepharose chromatography in a flow- through mode for IgG.

The optional step of nanofiltration of the IgG solution can be used to further reduce residual virus, in particular both enveloped and non-enveloped viruses. Preferably the filter has a nominal pore size of 20 nm to 35 nm.

U Itraf iltratio n/d iaf i Itration can be used adapt the buffer and to concentrate the product to the desired protein concentration. As mentioned in context with the pharmaceutical composition, a particularly desired concentration ranges between 90 and 1 10 g/L to decrease volume administered, thereby reducing the amount of excipients per treatment, The IgG solution can be formulated into a suitable pharmaceutical composition. This step can be achieved already by the previous step of ultrafiltration and diafiltration.

The pharmaceutical composition can be filled into a suitable container under sterile conditions. A suitable container can be a flask or a bottle suitable for infusion, e.g. comprising a sealed pierceable rubber stopper. Therefore, the present invention also relates to a container such as a flask or a bottle comprising the pharmaceutical composition. In addition, the present invention also relates to a package or kit comprising single or multiple flasks or bottles comprising the pharmaceutical composition alongside with instructions for administration (preferably with instructions for administration according to the administration scheme of the present invention).

The manufacturing process has been shown to be capable of inactivating or removing any potential blood-borne viral pathogens. The overall clearance capacity for enveloped and non-enveloped model viruses has been determined in the order of greater than 9 log₁₀ to greater than 12 log₁₀, and 5 log₁₀ to 10 logi₀, respectively."

The invention also provides for a preparation comprising a pool of hHCIG obtained from a pool of donors currently or previously infected with HCV. The content of such a hHCIG preparation can be controlled by an HCV-binding assay. Such an assay can be used to determine the anti-HCV antibody titer of the preparation required to be effective. Antibody release of the hHCIG preparation is based on standard Code of Federal Regulations criteria for immune globulins and the HCV antibody titer of the hHCIG preparation is obtained by testing with Ortho's Vitros ECi., or other commercial test kits. Typical values obtained by said assay lie in the range of at least 500 U/ml, e.g. between 500 to 2500 U/ml, or even higher.

The hHCIG preparation is HCV RNA negative, i.e. HCV-RNA should not be detectable in the hHCIG preparation. Starting from pooled HCV-positive plasma the process should be designed to achieve a theoretical aim of minus 5logi₀, preferably minus 6logi₀, i.e. a theoretical limit of 1 HCV RNA molecule in 6logi₀ bottles of the final preparation. This HCV- RNA measurement can be done using quantitative polymerase chain reaction (qPCR) such as for example the National Genetics Institute UltraQualt-1000 method (UltraQualT HCV RT- PCR assay), or other qPCR tests available from e.g. Roche. The hHCIG preparation according to the present invention can be formulated into a pharmaceutical composition. Preferably, the pharmaceutical composition according to the present invention comprises the hHCIG preparation as defined herein as a 4% to 12% solution, more preferably a 5 to 10%, most preferably a 10% +/- 1 % (90 - 1 10 g/L) hHCIG solution based on total immunoglobulin content. The concentration of 90 to 1 10 g/L has the advantage that the amount of excipients administered with each infusion is reduced compared to a lower concentration. Furthermore, the volume administered with each infusion is reduced compared to a lower concentration. This has been found to be of particular benefit to liver transplant patients, who receive multiple other medications and who do have a general risk of volume or liquid overload, either by other medication or by water retention after the transplantation. Preferably, the composition has physiological osmolality. In a preferred embodiment, the pharmaceutical composition according to the present invention is formulated in glycine, more preferably in about 300 mM glycine. Preferably, the formulation is buffered at a pH of 4 to 7, preferably of 4.0 to 4.6 in order to maintain a low dimer content, while a higher pH formulation (such as pH 5 to 6, e.g. about 5.2) will promote greater stability at room temperature storage in terms of IgG aggregates and fragments, preferably of about 4.3.

The pharmaceutical composition may additionally include or may be combined with other active ingredients suitable for liver transplantation, including suitable antivirals, anti- inflammatories, or immune modulators. The pharmaceutical composition may additionally include or may be combined with further active ingredients which are used to reduce the action of the immune system on transplanted cells, organs or tissues. The pharmaceutical composition may contain or be combined with substances which make the patient more comfortable during treatment, reduce side-effects, or enhance uptake. The pharmaceutical compositions may further comprise additives, excipients, or other substances which allow for more effective administration.

The terms "treat" or "treatment" encompass both the therapeutic treatment of an already developed disease or condition, as well as prophylactic or preventative measures to avoid disease recurrence, HCV infection or HCV re-infection. Beneficial or desired clinical results due to said treatment may include, without limitation: curing of the disease, alleviation of one or more symptoms of the disease, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, prevention of disease recurrence, amelioration or palliation of the disease state, and the like. "Treatment" may also mean prolonging survival as compared to expected survival if not receiving treatment. Specifically, the term treatment as used herein aims to cure the patient from HCV infection and/or liver disease and/or to avoid or diminish HCV-infection-induced disease and/or HCV re-infection in said patient.

The term "prophylactically effective amount" refers to an amount of the pharmaceutical composition that inhibits or delays in a subject the onset of HCV-infection, or liver disease in a subject. The term "therapeutically effective amount" as used herein, refers to an amount of pharmaceutical composition that elicits the biological or medicinal response in a subject that is being sought by a medical doctor or clinician, which may include inter alia alleviation of the symptoms of the disease or amelioration of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the pharmaceutical compositions defined herein. Each hHCIG infusion is minimally dosed at at least about 200 mg/kg, preferably of from 200 to 500mg/kg, more preferably of from 300 to 400 mg/kg, or a dose equivalent thereto, but can also be higher.

In the treatment method or scheme according to the present invention, said dosage can also be equivalent in antibody titer to the ones indicated herein. That is to say, if one would use a 5% hHCIG preparation, a double dosage would be needed in relation to the dosage of the 10% hHCIG preparation disclosed herein. Following the same reasoning, if one would use a 20% hHCIG preparation, only half of the dosage would be needed in relation to the dosage of the 10% hHCIG preparation disclosed herein.

The trough levels of hHCIG in subjects receiving treatment, i.e. the level of hHCIG in the blood of the subject under treatment should preferably be at least 100 U of anti HCV antibody per ml of tested patient blood sample. The examples section below shows that such a trough level is needed in order to reduce the HCV-RNA levels. This level is effectively reached in the patients treated with the method of treatment according to the present invention.

The assay(s) used for determining the anti HCV-antibody trough levels in the patient undergoing hHCIG treatment can be selected according to the knowledge of the skilled person. One possible method is an ELISA with a rubella antigen, and/or an ELISA employing HCV viral coat proteins, polymerases or core proteins. For example, the HCV Ab Titer-Ortho Vitros Eci (U/ml) and Rubella Ab Titer-Ortho Vitros Eci (lU/ml) kits can be used. Both are ELISA assays. Rubella is used as a control. Depending on the patient, there may also be endogenous levels of Rubella detectable.

In the context of the present invention, it has been found that the hHCIG preparation according to the present invention is capable of neutralizing residual HCV virus in patients having low or residual HCV titers such as patients undergoing liver transplantation or subjects acutely being exposed to HCV-positive blood.

In order to be effective and to reduce or eliminate HCV recurrence in case of liver transplantation, the hHCIG preparation should be administered surrounding and immediately |f||| transplantation together with a pretreatment with antiviral agents (prior to transplantation). Preferably, the hHCIG formulation is administered in a specific scheme comprising:

a) pre-treatment (i.e. before the liver transplant surgery (day 1 )) of the subject with antiviral agents (AV) reducing the viral load in the subject to HCV-RNA levels of ≤ 150, preferably≤ 100 infective units/ml (lU/ml); and

b) 16 total hHCIG infusions spread over a period of 10 weeks according to the following scheme:

b1 ) 3 infusions surrounding the liver transplant surgery (day 1 ), wherein at least the first dose is administered before transplant because HCV resides predominantly in the old liver and can enter from the old organ into the circulation of the subject. The second dose is administered during the surgical transplant and the third dose is administered following 6-8 hours after the end of the second dose.

b2) followed by 5 daily infusions on consecutive days;

b3) followed by 2 infusions twice per week (e.g. days 10, 14);

b4) followed by 4 infusions once per week (e.g. days 21 , 28, 35, and 42);

b5) followed by 2 infusions twice per month (e.g. days 56, and 70).

In step b1 ), the following treatment scheme is preferred: the first infusion is given before the liver transplant surgery, the second infusion is given during the anhepatic phase (when the diseased liver is removed), and the third infusion is given following 6 to 8 hours after said second infusion. This specific treatment scheme was based on prior HCIG studies and the PK modeling results. The loading dose just prior to transplantation was to allow "mopping" the viruses in circulation during the surgical event. Multiple doses in short intervals are provided to neutralize residual virus particles and maintain sufficient titers especially in the time of transplantation, blood loss and volume shifts.

The hHCIG treatment scheme according to the invention results in a very low HCV recurrence rate, especially in patients wherein the HCV virus was undetectable before liver transplant for over 90 days. In such patients, the HCV may still latently present, and can re- infect the newly transplanted liver. After treatment with the hHCIG treatment scheme according to the invention, said re-infection rate was almost absent. Each of the different steps in the scheme are further defined below.

The pre-treatment with antivirals (AV), intends to reduce the viral load to HCV RNA levels of < 100 infective units/ml (lU/ml). The duration of AV pre-treatment is not critical, as long as the threshold of HCV RNA < 100 lU/ml, preferably of < 43 lU/ml, more preferably of < 15 lU/ml is reached at the time of the liver transplant surgery. The duration of the pre-treatment will hence be patient-specific depending on the viral load. In case of high viral load (e.g. 10³ to 10⁶ lU/ml), the pre-treatment may be longer than for patients having a low HCV blood titer (e.g. below 500 lU/ml). In other words, a short pre-treatment (e.g. 1-10 days) can be sufficient if the viral load is sufficiently low in said patient. Thus, the time of pre-treatment with antivirals may be less than 12 weeks, less than 90 days, less than 60 days, less than 30 days or less than 15 days. The inventors however did observe that the lower the viral load at transplant is, the better the outcome of the treatment is. Hence, in preferred embodiments, the viral load should be below detection, i.e. below HCV RNA < 100 lU/ml, preferably≤ 50 lU/ml, more preferably≤ 43 lU/ml, even more preferably≤ 15 lU/ml. The antiviral treatment can be initiated up to 6 months prior to the liver transplant.

The HCV RNA presence can be determined by any quantitative method now in the art. One example is the commercially available COBAS® AmpliPrep/COBAS® TaqMan® HCV Test (Roche), which is an in vitro nucleic acid amplification test for the quantitation of hepatitis C RNA genotypes 1 to 6 in human EDTA plasma or serum and has a lower limit of quantification (LLOQ) of 45 lU/ml HCV-RNA (v.1.0) or 15 lU/ml HCV-RNA (v.2.0). The pre-treatment step or phase as used herein is defined as the procedural phase prior to the actual surgical liver transplantation. During said pre-treatment step, the subject will be treated with anti-viral agents (AV) in order to reduce the viral load in the subject to HCV-RNA levels of ≤ 100 infective units/ml (lU/ml). Said level needs to be reached prior to the liver transplantation procedure itself. Candidate antiviral agents can be selected from the group of: interferons (e.g. Intron A®, Rebetol®, Roferon®, Infergen®), pegylated interferons (e.g. Pegasys®, Pegintron®), ribavirin (CoPegus®), protease inhibitors (e.g. danoprevir, telaprevir, ACH-1625, ABT-450 HCV, VX-813, PHX1766, VX-500, GS-9256, Bl 201335, vaniprevir, narlaprevir, TMC 435 and boceprevir), polymerase inhibitors (e.g. RG7128, VX- 222, IDX375, ABT-072, MK-3281 , PSI-7851 , ABT-333, VX-916, filibuvir, IDX184, ANA598, GS9190, VX-759, and PSI-7977), NS5A inhibitors (e.g. BMS-790052, A-832, and PPI-461 ), NS4B inhibitors (e.g. clemizole), cyclophilin inhibitors (e.g. SCY-635 and Debio 025), TLR agonists (e.g. ANA773, IMO-2125, and SD-101 ), immunomodulators/interleukins (e.g. CYT 107, NOV-205, and oglufanide disodium), agents targeting liver-specific micro-RNA (e.g. SPC3649), A3AR agonists (e.g. CF102), anti-phospholipid therapies (e.g. bavituximab), inflammation inhibitors (e.g. CTS-1027), immune stimulants (e.g. SCV-07), thiazolides (e.g. nitazoxamide), monoclonal antibodies targeting HCV and pancaspase inhibitors (e.g. PF- 03491390).

Preferably, the antiviral agents used in the pre-treatment step are virostatics targeting NS3/4a, NS5a and NS5b, more preferably selected from the group comprising: Sofosbuvir, Telaprevir, Peginterferon, Ribavirin, Sofosbuvir, Simeprevir, Ledipasvir, Ombitasvir, Paritaprevir, Ritonavir, Dasabuvir, or combinations thereof. Particularly preferred combinations are Sofosbuvir and Ribavirin; Sofosbuvir and Simeprevir; Sofosbuvir, Ribavirin and Simeprevir; Telaprevir, Peginterferon and Ribavirin; Telaprevir and Peginterferon; Sofosbuvir, Peginterferon and Ribavirin, Sofosbuvir and Ledipasvir and a combination of Ombitasvir Paritaprevir Ritonavir and Dasabuvir.

The administration of the hHCIG preparation or pharmaceutical composition according to the invention can be administered via intravenous infusion. Preferably said infusion is done using a separate infusion line to avoid mixing with other drugs or medications that could interfere with the hHCIG preparation. Administration can be done in standard sodium chloride or dextrose saline solutions. Exemplary media can be 0.9% sodium chloride or 5% dextrose saline. Infusion rates can be chosen anywhere from 0.6 ml/kg/hr up to a maximum rate of 200 ml/hr.

In another aspect, the present invention provides for neutralizing residual HCV in the body of a subject. In a specific embodiment thereof, the invention provides for the treatment of a subject being acutely exposed to HCV-positive blood, such as through a needle-stick accident with an HCV contaminated needle or an accident with a contaminated surgical instrument, such as a lancet or dental instrument. In such subjects, only small amounts of HCV viruses enter in the circulation. Particularly, the viral load of such subject might not exceed a level of HCV RNA of ≤ 100 infective units/ml (lU/ml). A neutralization treatment with the hHCIG preparation, optionally in combination with known antivirals is therefore provided. An exemplary treatment scheme would entail at least one, two, or three infusions with the hHCIG preparation of pharmaceutical composition, during a period of approximately one week. For example, three consecutive daily infusions could be administered, or in case of a high contamination risk, more than one infusion could be given on the first day, to increase the hHCIG load in the subject. The treatment regimen should be started as soon as possible and preferably immediately after such exposure. A second infusion is to be given within 7 to 10 days from the 1^st infusion. If needed, additional hHCIG infusions can be administered.

In an alternative administration scheme for treating acute exposure to HCV-positive blood, one or more first hHCIG infusion(s) is(are) administered immediately after the exposure, a second infusion is administered on the next day, and a third infusion after about one week (6-8 days).

In yet another alternative administration scheme for treating acute exposure to HCV-positive blood, one or more first infusion(s) is(are) administered immediately after exposure and an additional infusion is administered on days 2 and 3. The HCV-RNA levels in the patient's blood can be measured regularly to monitor the response to the hHCIG preparation.

In yet another aspect, the present invention provides for preventing HCV transfer from mother to embryo, foetus or baby. This can be achieved by neutralization of HCV in circulation in the mother, preferably shortly before delivery, at which the risk of infection of the baby is highest. An exemplary treatment scheme would entail at least one, two, or three infusions with the hHCIG preparation of pharmaceutical composition, immediately prior to or during the delivery. For example, one, two or three consecutive infusions could be administered, depending on the viral load of the mother and the contamination risk. The risk of infection to the foetus from HCV infected mother is highest at the time of birth especially in case of high risk pregnancy (caesarean section, co-infection with HBV or HIV, etc.). The treatment scheme will typically include hHCIG treatment of the mother starting a few days or just prior to delivery and/or during delivery and optionally a second or further infusion to the baby within 24 hours after birth. A third or further infusion to the baby will be based on determination of viral loads and will be approximately within one week (6 to 8 days) of the second infusion. Depending on viral load in the mother, the administration prior to delivery can be increased in order to have a sufficiently high load of circulating anti-HCV antibodies, i.e. preferably reaching trough levels of at least 1 10 U of anti HCV antibodies per ml, preferably at least 120, 130, 140, 150, or 200 U anti HCV antibodies per ml. Alternatively, the mother could be treated prior to given birth, in order to achieve trough levels at least 1 10 U/ml of anti-HCV antibody, preferably at least 120, 130, 140, 150, or 200 U anti HCV antibodies per ml, before giving birth. This could lead to prophylaxis of the newborn.

In yet another aspect, the present invention provides for the use of hHCIG for the prevention of HCV-recurrence in re-transplant patients. An exemplary treatment scheme will be similar to the one of liver transplant patients as defined herein.

In yet another aspect, the present invention provides for the use of hHCIG for the prevention of HCV (re-)infection in transplantation of organs from HCV-positive donors, particularly of HCV-positive donors who had low viral loads, or had received antiviral treatments. A typical treatment scheme will be similar to the treatment scheme for liver transplant patients as defined herein, i.e. the scheme of 16 infusions over 10 weeks with prior antiviral treatment before re-transplantation.

In yet another aspect, the present invention provides for the use of hHCIG for the prevention of HCV (re-)infection during liver transplantation in HCV-negative recipients with donor organ of unknown or positive HCV status. A typical treatment scheme will be similar to the treatment scheme for liver transplant patients as defined herein, i.e. the scheme of 16 infusions over 10 weeks with prior antiviral treatment before re-transplantation.

The hHCIG preparation or pharmaceutical composition comprising the latter can further be defined in terms of immunoglobulin release in the subject. This so called trough level can be determined by analysis of a blood sample of the subject under treatment for anti-HCV antibodies. Comparing the anti-HCV antibody titer prior to hHCIG administration and post administration can give a good measurement of the actual antibody release invoked by the treatment scheme.

The dosage regimen of the hHCIG treatment has been set to at least 200 mg IgG /kg bodyweight of hHCIG preparation as defined herein, wherein the mg refers to the amount of total IgG by weight in the hHCIG preparation. In practice, the dosage regimen can be higher that said 200 mg/kg, and can e.g. be at least 300 mg/kg or can be about 300 mg/kg, at least 350 mg/kg, or about 350 mg/kg, at least 400 mg/kg, or about 400 mg/kg, at least 500 mg/kg, or about 500 mg/kg, or even higher. Also antibody doses equivalent to the doses mentioned herein would be applicable. In this respect, it should be clear that when a preparation is used with a higher concentration of hHCIG, a lower volume would be needed to achieve the same antibody dose and vice versa. Such equivalent doses are to be seen as being included within the scope of the present invention.

The hHCIG treatments according to the invention may be in parallel, subsequently, before or overlapping with a treatment with one or more antiviral agents according to the invention, in particular in overlapping or in parallel with a reduced dose of antiviral agent compared to a standard treatment with the antiviral agent.

The respective aspects of the invention as described herein will now be further illustrated by the following non-limiting examples.

All references cited herein are hereby incorporated by reference in their entirety.

Examples

Example 1 : Preparation of the hHCIG preparation according to the present invention

The hHCIG preparation as defined herein can be prepared using the following general process:

Step 1 : Thawing and pooling plasma

In this first step, stored frozen donor plasma is thawed at an air temperature between 30 and 40 °C to achieve a plasma temperature of 20-25°C. At the same time, the thawed plasma from different donors is randomly pooled in order to obtain the largest possible divergence in antibodies against HCV and in order to achieve an HCV antibody titer of about 500 U/ml. Typically about 300 plasma donations are needed for this. The pool calculated titer can be determined using the following formula: Pool calculated titer = (Unit Volume*Unit Titer) / Total Pool Volume.

The pool calculated titers of the plasma pools used as a starting product herein ranged between 505 and 530 U/ml, based on 314 to 379 donors per pool. The actual antibody titer of the pools was confirmed as lying between about 450-550 U/ml. The pool volumes were roughly between 1000 and 2000 liters.

A filtration step can be performed in parallel to the pooling process using e.g. filtration/clarification filters. Exemplary filtration/clarification filters are those having a pre-filter with nominal pore size of 1 μΐη. Plasma clarification is performed in order to reduce insoluble aggregates, thereby facilitating the upfront treatment with solvent/detergent and improving the efficiency of the virus reduction. The number of donors in the different lots was between 314 and 379.

Step 2: Upfront treatment with solvent/detergent

Since the donor blood plasma is HCV positive, an upfront viral inactivation step is very important. In this first virus inactivation step, Tri-n-butyl phosphate (TnBP) solution is added to the pooled plasma to achieve a final concentration of 1.1 % w/w and Triton X-100 solution is added to a final concentration of 1.1 % w/w while stirring. This mixture is stirred at a temperature of about 30°C (± 2°C) for a minimum of 4 hours. The virus inactivated plasma is then transferred into a reactor tank for the following ethanol precipitation steps.

Step 3: Precipitation/separation of Fraction I

The S/D treated plasma solution is adjusted to about pH 7.3 ± 0.1. Subsequently, cold SDA- 3A alcohol (denatured ethanol) is slowly added to a final total alcohol concentration of 8 ± 3% by volume with gentle mixing and simultaneously the temperature is slowly lowered to -2 ± 1 °C. Under continuous mixing during and after the alcohol addition, the Fraction I precipitate forms. Fraction I precipitate contains several plasma derived components such as fibrinogen and other clotting factors and is discarded. Fraction I supernatant is further processed to Fraction ll/lll.

Step 4: Precipitation/separation of Fraction ll/lll

The pH of the Fraction I centrifugate is adjusted to pH 6.9 ± 0.1 Then cold ethanol is added under continuous mixing to a final alcohol concentration of 25 ± 3% by volume and simultaneously the product temperature is slowly lowered to -5 ± 1 °C. Fraction ll/lll is separated by centrifugation to collect the precipitate fraction which is further processed.

Step 5: Resuspension of Fraction ll/lll

For further processing, Fraction ll/lll (see figure 3.2. S.2.2-2) is resuspended in 2 volumes of Water For Injection (WFI) then buffer (e.g. phosphate) at 1 ± 1 °C for upto3 hours. Step 6: Precipitation/separation of Fraction ll/lllw and pooling

In order to precipitate Fraction ll/lllw, the pH of the suspension is adjusted to 7.20 ± 0.1 For every kilogram of Fraction ll/lll precipitate, twenty volumes of cooled WFI are added to the Fraction ll/lllw suspension. Subsequently cold ethanol is added under constant mixing to a final concentration of 20 ± 3% by volume and simultaneously the temperature is slowly lowered to -5 ± 1 °C.

Fraction ll/lllw is separated by centrifugation (e.g. using Pennwalt type Clarifier Centrifuge Model No. AS26), while maintaining the product temperature at -5 ± 1 °C. The intermediate, Fraction ll/lllw paste is removed from the bowls and is either processed. Fraction ll/lllw precipitate (paste) is weighed and resuspended in WFI at 1 ± 1 °C and stirred for a minimum of 3 hours. Then further diluted with WFI and Sodium Acetate solution while maintaining a suspension temperature of at or below 2°C.

Step 7: Precipitation/separation of Fraction III

The pH of the Fraction ll/lllw precipitate suspension is adjusted to pH 5.30 ± 0.1. Then cold ethanol is added under stirring to a final alcohol concentration of 17 ± 3% by volume and simultaneously the temperature is slowly lowered to -5 ± 1 °C.

Fraction III is separated by centrifugation (e.g. using a Pennwalt type Clarifier Centrifuge Model No. AS26) while maintaining the product temperature at -5 ± 1 °C. The intermediate, Fraction III paste is removed from the centrifuge bowls and is discarded. The Fraction III supernatant is further processed.

Step 8: Depth filtration of Fraction III centrifugate

Fraction III supernatant (0.5 is clarified by the depth filtration at a temperature of -5 ± 1 °C The quality of the filtration is verified by an in-process turbidity measurement. The Fraction III filtrate is further processed.

Step 9: Ultrafltration/diafiltration of Fraction III filtrate

The pH value of the fraction III filtrate is slowly adjusted to 4.1 ± 0.1 continuous stirring while maintaining the temperature at -5 ± 1 °C.

The protein solution is concentrated to a protein content of approximately 70 g/L and subsequently diafiltered against a quantity of WFI to remove residual ethanol from the previous precipitation steps.

Step 10: Treatment with solvent/detergent

Tri-n-butyl phosphate (TnBP) solution is added to the protein solution to achieve a final concentration of 0.2-0.4% w/w and 20% Triton X-100 solution is added to a final concentration of 0.8-1.2% w/w while stirring. The protein solution is stirred at a temperature of 28 ± 2°C for a period of at least 2 hours.

Step 11 : Column chromatography Two chromatography steps are used in order to further purify the IgG product. Firstly, a reversed phase C-18 gel is used for the extraction of the SD reagents. While the SD reagents are bound to the C18 matrix the protein solution flows through the column. The column is eluted with WFI at 15-30°C. The protein solution is collected until the absorbance is < 0.2 AU. The pH is adjusted to 7.5 ± 0.2 and conductivity is checked (specification 1 1.5 ± 1 mS). Next, Q- Sepharose Column chromatography is performed to remove trace levels of plasma derived impurities such as IgA, IgM, and PKA. The C18 eluate is pumped over the column at a flow rate of 5.0 L/min. The column is eluted with phosphate buffer at 15-30°C and the protein solution is collected until the absorbance is < 0.2 AU. The pH is adjusted to 4.2 ± 0.1.

Step 12: Nanofiltration (20-35nm filter)

The chromatography purified IgG solution (< 25 g/L) is filtered through nanofilters with a nominal pore size of 20-35 nm (e.g. Asahi, Planova filters).

Step 13: Ultrafltration/diafiltration The obtained protein solution is subsequently concentrated using 10,000 Dalton UF- membranes to a protein content of 100-120 g/L. The solution is subsequently diafiltered against 8 times the protein solution volume with final formulation glycine buffer of 300 mM.

Step 14: Formulation of the hHCIG preparation in glycine

The protein solution is adjusted to a calculated protein content of 100 g/L 110 g/L with final formulation buffer and the pH is adjusted to 4.3 (important to avoid antibody dimer formation). The resulting hHCIG solution is filtered through a 0.2 μιη filter into sterile containers and the obtained hHCIG preparation is stored at 5 ± 3°C until use. The final characteristics of the hHCIG preparation are given in Table 1 below.

Table 1 : The final product had the following characteristics:

Analytical Average values for hHCIG

Formulated Bulk

Method lots

Complement

ACA fixation (Ph. Eur) 0.98 mg lgG/ml/CH50

Protein Biuret 97.2 g/L Total IgG Nephalometry 99.3 mg/ml

HPLC (size

Aggregates exclusion) 0

HPLC (size

Fragments exclusion) 0

HPLC (size

Monomer+Dimer exclusion) 100%

Amidolytic Ph.

Prekallikrein Eur. < 3.64 % Ref. Std.

Gas

chromatography

TnBP (FID detection) < 0.3 ppm

HPLC (reverse

Triton X-100 phase) < 1.5 ppm

It is important to see that the hHCIG preparation is concentrated for IgG antibodies and contains little or no IgA and IgM antibodies.

The final preparation was also tested for residual HCV RNA presence determined by the National Genetics Institute UltraQualt-1000 qualitative method and need to be negative for HCV RNA (< level of detection 5 copies/ml).

The anti-HCV antibody titer was measured using the Vitros assay (cf. Table 2 below). The results were the following for 6 different lots prepared:

Table 2:

PotencyTiter

Lot # (U/ml)

1 1882.89

2 1880.67

3 2254.82

4 2410.55

5 2193.58

6 2175.72 These measurements were made based on a 10% immunoglobulin solution, comprising on average between 90 and 1 10 mg/ml protein, wherein at least 95% is IgG protein. The final preparations easily obtained an Anti-HCV antibody titer of between 1800 and 2500 U/ml.

Example 2: Clinical trial results of hHCIG preparation treatment schedule of the invention

A Multi-Center, Randomized, Prospective, Open-Label Phase III Study was performed using the hHCIG preparation as prepared and formulated in Example 1 , to evaluate the efficacy, safety and pharmacokinetics thereof in orthotopic Liver Transplant recipients. The study is ongoing and the results herein are those obtained at the current date.

Patient characteristics

In this section an overview is given of the patient's characteristics. The main characteristics are given in table 3 below:

Table 3a:

Table 3b: Group N MELD (Median) CP-Class A (%) CP-Class B (%) CP-Class C (%)

Control 22 19 35.0 30.0 35.0

200 mg/kg 20 1 1.5 55.6 27.8 16.7

300 mg/kg 21 24 37.5 37.5 25.0

All 63 16.5 42.6 31.5 25.9

N= number of patients; CP = Child-Pugh

Patient inclusion criteria:

In general the patients needed to comply with the following criteria in order to be selected for the clinical trial:

1. Written informed consent obtained prior to any study-specific assessments and within 3 months (reconsent) of orthotopic liver transplantation (OLT).

2. HCV Genotype 1- 6 Infection.

3. Subjects in the beginning of a new antiviral therapy regimen (regardless of prior treatment failures) for up to and including 24 weeks prior to the day of OLT and not having achieved Sustained Virological Response 12 weeks after end of antiviral treatment (SVR12).

4. Most recent PCR test within the last 4 weeks that HCV RNA is <100 lU/ml. Subjects may be randomized based on local lab HCV RNA results if central lab results are not available at time of randomization.

5. Male and female subjects (age 18 to 80 years of age).

6. Stable Subject in a condition which in the opinion of the investigator would permit safe participation in the study.

Patient exclusion criteria:

Patients were excluded from the clinical trial in case they complied with the following criteria: 1. Re-transplantation due to viral recurrence. 2. Positive HIV test or HBV positive surface antigen test within 90 days prior to transplantation.

3. Most recent PCR test indicating HCV RNA≥100 lU/mL within 4 weeks of OLT.

4. Subjects having received organs from HCV positive donors.

5. Serum creatinine level >2.5 times the upper limit of normal or advanced renal disease at screening.

6. Pregnancy or single contraceptive measure or lactation period(females only).

7. Known intolerance to immunoglobulins or comparable substances (e.g. vaccination reaction).

8. Known absolute IgA deficiency.

9. Known intolerance to proteins of human origin.

10. Participation in another clinical trial within 90 days before signing Informed Consent Form (ICF) or during the study (observational/ non-interventional and 988 sub-studies allowed), and/or previous participation in 988 study (except for Study 988 screen failures). 11. Active drug and/or alcohol abuse.

12. Inability or lacking motivation to participate in the study.

Genotype representation in patients

The HCV genotype differed amongst the patients in the group, but type 1 (including types 1 a and 1 b) was predominantly present. An overview is given in table 4 below.

Table 4: Genotype representation in patients:

Type 1* 13%

Type 1a 59%

Type 1 b 21 %

Type 2 2% Type 3 5%

Others** 5%

^* Type 1 can encompass both Types 1 a and 1 b, since the sub-genotype was not determined *^* Genotype not known

The Median HCV RNA pre-antiviral therapy in all patients was 8 weeks. The following antiviral agents were used:

Antiviral pre LT -treatments

Different antiviral treatment schemes have been used in different patients. Table 5 below gives an overview of the treatments used.

Table 5:

Sofosbuvir, SIM= Simeprevir

In order to assess the influence of said antiviral treatment, the HCV-reinfection was compared between the different antiviral treatment schemes. As shown in the three tables 7 to 9 below (resp. control group, 200mg/kg group and 300 mg/kg group), there is no correlation between the type of antiviral treatment and the HCV-reinfection risk. Dosage Regimen

The dosing regimen consists of 16 infusions over a 10 week period that starts just prior to the OLT as listed below. HCIG was administered three times surrounding surgery: once beginning before, once at start of or during the anhepatic phase and once after OLT. Daily infusions occurred through Day 6. Subsequent infusions occurred on Days 10, 14, 21 , 28, 35, 42, 56, and 70. Apart from HCIG doses #1 through #3, reasonable effort was made to administer HCIG in the morning (cf. Table 6).

Table 6: The following treatment scheme was typically applied:

Dose Timing Doses Dose #

Pre-transplant Dose (18 to 3

200/300mg/kg

hrs before hepatectomy)

Start of anhepatic phase 200/300 mg/kg

Post-transplant dose^*

(6-8 hrs after anhepatic dose) 200/300 mg/kg

Day 1

Day 2 200/300 mg/kg

Day 3 200/300 mg/kg

Day 4 200/300 mg/kg

CO

Q

Day 5 200/300 mg/kg

Day 6 200/300 mg/kg

Day 10 200/300 mg/kg

Day 14

X

CM 200/300mg/kg 10

(end week 2)

Day 21

200/300 mg/kg 11

X (end week 3)

^* The first three infusions are given around the time of surgery and their administration scheme can differ due to the surgical timing or set-up. Infusion 4, i.e. after transplantation is generally given in the morning, regardless of the time of transplant (e.g. morning, midday, evening, midnight). This implies that the time between administering infusions 3 and 4can be anywhere from e.g. 6 to 24 hours.

Dosage calculation and Administration

Dose Calculation

All doses are based on the recorded hospital admission weight, prior to transplant. The calculated total dose for all infusions was based on the admission weight, and was recorded for each dose.

Preparation of Study Medication

HCIG as prepared in Example 1 was brought to room temperature for approximately 15 minutes or more before administration and used within 10 hours of removal from refrigeration. Multiple vials may be pooled under aseptic conditions into an individual infusion bag and hung directly with the infusion set.

Administration

HCIG was infused intravenously via an infusion pump using a separate infusion line. Care was taken not to mix with other drugs or medications. Based on compatibility experience with other globulin preparations, HCIG was infused sequentially into an IV line containing either 0.9% sodium chloride or 5% dextrose saline. The initial infusion rate was be 0.01 imL/kg/minute (0.6 mL/kg/hr). In the absence of adverse events, the infusion rate was increased by 0.01 mL/kg/min (0.6 mL/kg/hr) every 20 minutes to a maximum of 0.06 mL/kg/min (3.6 mL/kg/hr) up to a maximum rate of 200 mL/hr. The concentration of HCIG is 100 mg protein/ml. Subsequent infusions were administered at the optimum rate or at a rate determined by the investigator. However, the maximum rate of 200 ml/hr was never exceeded.

Treatment results

In this section, the treatment results to this date are described for the control group receiving a placebo, for the 200mg/kg group and for the 300mg/kg group. The following abbreviations are used throughout tables 7 to 9:

meg microgram

SC subcutaneous

BID twice daily

TID three times a day

QAM every day before noon

QPM every day after noon / evening

QD every day

IV intravenous

HCC Hepatocellular Carcinoma

UND Undetectable (defined as below 43 lU/ml)

TOR Transplantation and Randomization

LT Liver transplantation

Duration Days between start and stop date of antiviral treatment

Days UND prior

LT Days between LT and earliest undetectable PCR result that was measured

0 = Patient was undetectable or below 100 lU/ml prior to transplantation at the day of transplantation and no prior negative PCR result was available.

Days to become Defined as difference of days between start of AV treatment and first UND PCR

UND result

Viral RNA at

TOR < 43 = Viral RNA detectable, but below limit of quantification (< 43 lU/ml) 51

Figure 3: represents the status of control group patients. The complete results are given in Table 7 below.

Table 7: Patient characteristics of the control group

Control Group

Sofosbuvir 400 mg QD: ORAL 117 0 117 Target Not Detected HCC no

Ribavirin 1200 mg QD: ORAL 1 16

Sofosbuvir 400 mg QD: ORAL 57 0 57 Target Not Detected HCC yes

Ribavirin 300 mg BID: ORAL 57

Ribavirin 600 mg BID: ORAL 88 0 < 43 HCC yes

Sofosbuvir 400 mg QD: ORAL 88

Any

Pegylated ONCE PER

interferon 135 meg WEEK: SC 49

Any

Pegylated 18,000,000 ONCE PER

interferon IU WEEK: SC 7

Ribavirin 600 mg QD: ORAL 17 < 43 no

Ribavirin 400 mg QD: ORAL 17

Sofosbuvir 400 mg QD: IV 17

Ribavirin 200 mg QD: ORAL 125 90 36 Target Not Detected HCC yes

Sofosbuvir 400 mg QD: ORAL 125

Ribavirin 1000 mg QD: ORAL 127 0 127 Target Not Detected HCC no

Sofosbuvir 400 mg QD: ORAL 127

600-400

mg (28)- mg (28)

Ribavirin DsPk mg BID: ORAL 64 HCC yes

Sofosbuvir 400 mg QD: ORAL 54

Sofosbuvir 400 mg QD: ORAL 65 1 64 Target Not Detected no

Simeprevir 150 mg QD: ORAL 65

Simeprevir 150 mg QD: ORAL 13 Target Not Detected no

Sofosbuvir 400 mg QD: ORAL

Ribavirin 600 mg BID: ORAL 0 Target Not Detected HCC no 53

Figure 4: represents the status of the 200 mg/kg group patients. The complete results are given in Table 8 below. Table 8: Patient characteristics of the 200 mg/kg group

200 mg/kg Group

Boceprevir 800 mg TID: ORAL 102 < 43 no

Any

Pegylated ONCE PER

interferon 180 meg WEEK: SC 102

Target Not

Ribavirin 1200 mg QD: ORAL 113 50 63 Detected HCC yes

Sofosbuvir 400 mg QD: ORAL 113

once per Target Not

Sofosbuvir 400 mg day: ORAL 44 12 32 Detected HCC no

Ribavirin 200 mg TID: ORAL 44

Any

Pegylated ONCE PER

interferon 180 ug WEEK: SC 44

Target Not

Sofosbuvir 400 mg QD: ORAL 47 16 31 Detected no

Ribavirin 200 mg QD: ORAL 32

Ribavirin 400 mg BID: ORAL 13

Ribavirin 400 mg QD: ORAL 0

Ribavirin 200 mg QD: ORAL 0

Ribavirin 600 mg BID: ORAL 36 < 43 HCC yes

Sofosbuvir 400 mg QD: ORAL 36

Target Not

Ribavirin 400 mg BID: ORAL 21 0 21 Detected no

Simeprevir 150 mg QD: ORAL 21

Sofosbuvir 400 mg QD: ORAL 21

Target Not

Ribavirin 600 mg QD: ORAL 115 Detected no

Sofosbuvir 400 mg QD: ORAL 1 15

Target Not

Ribavirin 300 mg BID: ORAL 70 0 69 Detected HCC no

Sofosbuvir 400 mg QD: ORAL 70 Any

Pegylated 180 ONCE PER

interferon mcg/ml WEEK: SC < 43 HCC yes

Ribavirin 400 mg BID: ORAL

Sofosbuvir 400 mg QD: ORAL

Target Not

Sofosbuvir 400 mg QD: ORAL 67 0 Detected HCC no

Ribavirin 600 mg QD: ORAL 42

Ribavirin 400 mg QPM: ORAL 42

Simeprevir 150 mg QD: ORAL 26

Target Not

Ribavirin 800 mg QD: ORAL 0 Detected HCC yes

Sofosbuvir 400 mg QD: ORAL

Ribavirin 500 mg BID: ORAL 54 0 54 Cancelled HCC no

Sofosbuvir 400 mg QD: ORAL 54

Simeprevir 150 mg QD: ORAL 30 0 30 < 43 no

Sofosbuvir 400 mg QD: ORAL 30

Target Not

Sofosbuvir 400 mg QD: ORAL 62 0 62 Detected HCC no

Simeprevir 150 mg QD: ORAL 62

Sofosbuvir 400 mg QD: ORAL 83 88520 HCC no

150

Simeprevir Capsule QD: ORAL 83

Target Not

Sofosbuvir 400 mg QD: ORAL 133 0 Detected HCC no

Ribavirin 1000 mg QD: ORAL 133

Sofosbuvir 400 mg QD: ORAL 35 < 43 HCC yes

Ribavirin 200 mg BID: ORAL 35

Target Not

Sofosbuvir 400 mg QD: ORAL 114 0 Detected HCC no 56

Figure 5 represents the status of the 300 mg/kg group patients. The complete results are given in Table 9 below.

Table 9: Patient characteristics of the 300 mg/kg group

300 mg/kg Group

interferon WEEK: SC

Median all Treatment Durations 49

Median Longest Treatment Duration per

Patient 69

Median Sofosbuvir Treatment Duration 60

Efficacy of the treatment schedule at data cut-off:

In Table 10 below, the overall efficiency of the treatment in the trial until today are represented:

Safety and adverse effects at data cut-off:

In Table 1 1 below, the overall adverse effects observed in the trial until today are represented:

None of the severe adverse events (SEA's) were related to the treatment. One has to keep in mind that the patients undergoing liver transplant are of course seriously ill. Some adverse events (AE) such as nausea, shoulder pain, fever, anemia, were observed in a minority of the subjects undergoing hHCIG treatment in both tested dosage groups. No dose limiting toxicities, no pregnancies, and no deaths attributable to the hHCIG were reported during the study.

Example 3: Ortho VITROS® ECiQ Immunodiagnostic System, Cat No. 680 1893

In principle, the test methods use recombinant peptides encoded by the Hepatitis C Virus:

In detail, the system uses three recombinant hepatitis C virus encoded antigens (c22-3, c200 and NS5). The recombinant protein c22-3 is encoded by the putative core region of the HCV genome. The HCV recombinant protein c200 is encoded by the putative NS3 and NS4 regions of the HCV genome and contains the c33c protein sequence which is genetically linked to the c100-3 protein sequence. Additionally, the assay makes use of the HCV recombinant protein NS5.

A two-stage immunometric technique is used. In the first stage, HCV antibody present in the sample binds with recombinant HCV antigens coated on the wells. Unbound sample is removed by a wash step.

In the second stage, a horseradish peroxidase (HRP)-labeled antibody conjugate is added which binds to any human antibodies. Any unbound conjugate is washed off followed by addition of a luminogenic substrate. This substrate is a combination of a luminol derivative, peracid salt and an electron transfer agent. The HRP in the antibody conjugate catalyzes the oxidation of the luminol derivative resulting in a light signal. The electron transfer agent increases the level of light produced and prolongs its emission. The light produced is measured in adjusted light units (ALUs). The amount of light generated is directly proportional to the concentration of the element in a testing solution.

Vitros ECiQ Anti-HCV components are stored and handled according to the package insert. In order to reduce the possibility of non-specific binding, the pre-coated wells are manually removed from the Vitros HCV reagent packs and manually pre-blocked using a blocking solution. The blocked wells are incubated overnight then washed in order to remove excess blocking solution. The blocked wells are loaded manually back into the Vitros HCV reagent packs which are then loaded onto the Vitros ECi instrument for automated analysis of the assay using chemiluminesence to measure the amount of light produced from the reaction in adjusted light units (ALUs). The raw data (ALUs) obtained from the ECi instrument is downloaded using a floppy disc onto excel. The desired data (sample identification and ALUs) is copy and pasted onto the JMP software for ANOVA and regression analysis. The software is used to convert ALUs into U/ml using a five point parameter calibration curve. The curve consists of six points made up from dilutions of a known reference standard. Potency results for the samples are reported in U/ml. The overall mean potency of the samples is calculated using a minimum of three valid sample dilutions (each tested in triplicate). Using the test, the kit's positive control should have a "Mean ALU of equal or greater than 1000 U/ml. A negative control, such as a commercially available IVIG derived from HCV negative donors, should have an ALU of equal or less than 50 U/ml.

The reference standard can be determined by measuring the titers in blood plasma of a suitable random sample of HCV-RNA positive subjects, particularly at least 50 subjects, more particularly at least 80, more particularly at least 150 subjects. Next, the anti-HCV antibody titers of the subjects in said sample are measured. The titers of the 30% subjects with the lowest titer and the titers of 30% of subjects with the highest titer are then excluded from further analysis, so that a subgroup of titers is formed. Said average titer is then calculated as the arythmetic mean of the titers of said subgroup. Said average titer obtained by this method is defined as 500 U/ml of blood plasma.

Example 4: Determining the minimum effective concentration of hHCIG composition

The minimum effective concentration, MEC, (HCV Ab titer) is estimated to be approximately 110 U/ml. This estimate is derived from evaluating HCV Ab titers over time and correlating them with the time of HCV-reinfection.

Total anti-HCV antibody concentrations were plotted over time for each subject on a semilog scale to visually assess their disposition profile. Initial pharmacokinetic parameter (Tmax, Cmax, AUCO-t, AUC0-∞, t1/2) estimates for each subject on each pharmacokinetic day (Study Day 6, 14, and 42) were determined by non-compartmental analysis. The elimination rate constant was estimated if a given subject had > 2 concentration values in the terminal portion of the curve. From these initial estimates and any correction for underlying endogenous levels of the antibodies, the data from each subject was modeled using an infusion model with first-order elimination. One compartmental body model was used to best describe the post-infusion disposition profiles of each antibody.

Figure 7a shows the median HCV Ab titers in Control Group subjects that reinfected and subjects which are viral free. For those subjects with reinfection, there was a relatively quick decline in HCV Ab titers post transplant below 1 10 U/ml. For the viral free subjects, the HCV Ab titers were predominantly above 1 10 U/ml throughout the course of follow up. Figure 7b shows the individual and median baseline HCV Ab levels per treatment and reinfection (groups with reinfections are marked with (R) in the figure).

The trends on HCV antibody (Ab) titers over time and the pharmacokinetic profile of hHCIG treatment were evaluated. Figures 7c to 7f show the correlation of viral load assessed by HCV RNA with HCV Ab titers in 4 different patients. At the time of the data cut-off for PK data evaluation, there were no reinfections for subjects in the 300 mg/kg dose group. In the 200 mg/kg group, 3 out of 4 subjects that were reinfected had HCV antibody titers drop below 1 10 U/ml (See Figures 7d to 7f). For these 3 subjects, each showed a marked increase in HCV RNA (viral load) within a study visit of the drop to or below 110 U/ml. This pattern of drop of ab levels below MEC in reinfected patients is consistent with that observed in the subjects not treated with hHCIG (Control group). The extent and time above the MEC may be a determining factor in preventing reinfection. Subjects with Ab titers near the MEC may be at more risk of reinfection, especially during times of increased interval between infusions.

Claims

1. A purified human hepatitis C immunoglobulin (hHCIG) preparation, isolated from pooled blood plasma substantially obtained from at least 200 different HCV antibody positive donors, wherein the anti-HCV-antibody titer of each donor individual is at least 10 U/ml, preferably at least 15 U/ml, more preferably at least 20 U/ml, most preferably at least 25 U/ml and wherein the anti-HCV antibody titer of the pooled plasma is at least 400 U/ml, preferably at least 500 U/ml, or a pharmaceutical composition comprising such a hHCIG preparation.

2. The pharmaceutical composition according to claim 1 , characterized in that the number of HCV antibody positive donors it at least 250, more preferably at least 300.

3. The pharmaceutical composition according to claim 1 , characterized in that the number of HCV antibody positive donors is not higher than 1000, particularly not more than 900, more particularly not more than 800, more particularly not more than 600 different donors.

4. The pharmaceutical composition according to claim 1 , characterized in that the HCV antibody positive donor group comprises both low and high HCV-antibody titer donors, preferably wherein the HCV-antibody titer of said high titer donors is at least 40 fold higher than the titer of the low titer donors, preferably at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least 100 fold higher.

5. The pharmaceutical composition according to claim 1 , characterized in that it comprises a formulation of a 90 to 1 10g/L immunoglobulin solution formulated in between 230 and 320 mM, preferably in about 300 mM glycine.

6. The pharmaceutical composition according to claim 1 , characterized in that it is formulated at a pH of between 4.0 and 4.6, preferably of about 4.3.

7. The pharmaceutical composition according to claim 1 , characterized in that the plasma from which it is derived is HBV-DNA, HAV-RNA, and HIV-RNA non-reactive (negative) and Parvo B19 DNA levels do not exceed 1 x 10³ lU/ml.

8. The pharmaceutical composition according to claim 1 , characterized in that the plasma from which it is derived is negative for Hepatitis B Surface Antigen (HBsAg), and HIV 1 , and HIV 2 antibodies.

9. The pharmaceutical composition according to claim 1 , characterized in that it is HCV- RNA negative.

10. The hHCIG-comprising pharmaceutical composition according to claim 1 , obtained from blood plasma of donors that have ALT-levels not higher than twice the upper level of the established normal range, preferably from blood plasma of donors that have ALT-levels below 219 U/ml, more preferably of below 150 U/ml.

1 1. The hHCIG-comprising pharmaceutical composition according to claim 1 , wherein said HCV virus is of anyone of genotypes 1 to 6, more preferably of genotypes 1 , 2, or 3.

12. The hHCIG-comprising pharmaceutical composition according to claim 1 , having an

HCV antibody titer of more than 1000 U/ml, preferably of between 1000 and 4000 U/ml, preferably of between 1500 and 3000 U/ml.

13. A method of treating HCV infection in a subject in need thereof comprising the administration of a dose of at least about 200 mg IgG /kg, preferably of from 200 to 500 mg IgG /kg, more preferably of from 300 to 400 mg IgG /kg, or a dose equivalent thereto of a hHCIG-comprising pharmaceutical composition, preferably the hHCIG- comprising therapeutic composition according to claim 1 to said subject.

14. The method according to claim 13, wherein the dose of said hHCIG-comprising therapeutic composition results in trough levels of hHCIG in subjects receiving treatment of at least 1 10 U of anti HCV antibodies per ml , preferably of at least 120,

130, 104, 150, or 200 U of anti HCV antibodies per ml.

15. The method according to claim 13, wherein said hHCIG-comprising composition is administered according to the following treatment regimen:

c) pre-treatment before the liver transplant surgery of the subject with anti-viral agents (AV) reducing the viral load in the subject to HCV-RNA levels of < 100 infective units/ml (lU/ml) prior to the liver transplantation,

d) at least 12, preferably 14 to 20 infusions of a hHCIG-comprising pharmaceutical composition, preferably the hHCIG-comprising therapeutic composition according to claim 1 to said subject, spread over a period of 6 to 15 weeks, comprising the following steps: b1 ) 2 to 4 infusions, preferably 3 infusions, surrounding the liver transplant surgery (day 1 )

b2) at least 9 infusions, preferably at least 12 infusions, spread over 6 to 15 weeks, preferably starting with daily intervals, preferably wherein the amount of each infusion is about 200 to 500 mg IgG per kg body weight.

16. The method of treatment according to claim 13, wherein said hHCIG-comprising composition is administered according to the following treatment regimen:

e) pre-treatment (i.e. before the liver transplant surgery) of the subject with anti-viral agents (AV) reducing the viral load in the subject to HCV-RNA levels of ≤ 100 infective units/ml (lU/ml), prior to the liver transplantation.

f) 16 infusions of the hHCIG-comprising therapeutic composition spread over a period of 10 weeks according to the following scheme:

b1 ) 3 infusions surrounding the liver transplant surgery (day 1 ), wherein at least the first dose is administered before transplant because HCV resides predominantly in the old liver and can enter from the old organ into the circulation of the subject;

b2) followed by 5 infusions on consecutive days (days 2, 3, 4, 5, and 6);

b3) followed by 2 infusions twice per week (e.g. days 10, 14);

b4) followed by 4 infusions once per week (e.g. days 21 , 28, 35, and 42);

b5) followed by 2 infusions twice per month (e.g. days 56, and 70).

17. The method of treatment according to claim 13, wherein the antiviral are virostatics targeting NS3/4a, NS5a and/or NS5b, preferably selected from the group comprising: Sofosbuvir, Telaprevir, Peginterferon, Ribavirin, Sofosbuvir, Simeprevir, or combinations thereof.

18. The method of treatment according to claim 16, wherein said reduced viral load is obtained at least 4 weeks prior to the liver transplantation and maintained at said reduced viral load.

19. The method of treatment according to claim 13, wherein the antiviral treatment is initiated up to 6 months prior to the liver transplantation.

20. The method of treatment according to claim 13, wherein said reduced viral load is≤ 50 lU/ml, more preferably≤ 43 lU/ml prior to the liver transplantation.

21. The method of treatment according to claim 13, wherein said patient is a subject infected with HCV, more specifically with HCV genotype 1 , 2, or 3, more specifically HCV genotype 1.

22. The method of treatment according to claim 13, wherein said patient has chronic liver disease, liver cirrhosis, (strongly) decompensated liver cirrhosis, or liver cancer.

23. A method for neutralizing residual HCV in the body of a subject comprising the administration of the hHCIG-comprising pharmaceutical composition according to claim 1 , wherein said treatment is optionally combined with other antiviral agents.

24. A method of treating a subject having been acutely exposed to HCV-positive blood, comprising the administration of the hHCIG-comprising pharmaceutical composition according to claim 1 , wherein said treatment is optionally combined with other antiviral agents.

25. A method for preventing HCV transfer from mother to embryo, foetus or baby comprising the administration of the hHCIG-comprising pharmaceutical composition according to claim 1 to the mother, wherein said treatment is optionally combined with other antiviral agents.

26. A method of preventing HCV-recurrence in re-transplant patients, comprising the administration of the hHCIG-comprising pharmaceutical composition according to claim 1 to the patient, wherein said treatment is optionally combined with other antiviral agents.

27. A method of preventing HCV (re-)infection in a patient undergoing transplantation of a donor organ of unknown or positive HCV status, comprising the administration of the hHCIG-comprising pharmaceutical composition according to claim 1 to the patient, wherein said treatment is optionally combined with other antiviral agents.

28. A method for producing an hHCIG-comprising preparation comprises the steps of: a) thawing of frozen plasma comprising anti-HCV antibodies, preferably at 20-40 °C, b) reduction of insoluble aggregates from the thawed plasma,

c) treatment of the plasma depleted of insoluble aggregates with solvent/detergent, d) isolating, preferably by ethanol fractionation, from the solvent/detergent treated plasma an IgG containing solution comprising at least 93%, preferably at least 95%, most preferably at least 98% pure IgG based on the total protein content, e) optionally, further clarification of the IgG containing solution,

g) treatment with a solvent/detergent solution,

h) reduction of the solvent/detergent from the solution of step g),

k) nanofiltration of the IgG solution,

I) ultrafiltration and diafiltration to the IgG-containing solution,

m) optionally formulating said hHCIG preparation into a pharmaceutical composition, and

n) optionally the pharmaceutical composition is filled into a suitable container.

29. A container suitable for infusion comprising the hHCIG preparation or pharmaceutical composition according to claim 1.

30. A package or kit comprising a single or multiple container(s) according to claim 28, and instructions for administration, preferably with instructions for administration according to the administration scheme defined in claim 15.