Method for polyclonal immunoglobulin g production by human b cells

Abstract

This application relates to an in vitro method of producing a polyclonal IgG preparation. The method comprises (i) placing a polyclonal B-cell population enriched in IgG-secreting B cells in a culture medium; and (ii) culturing the polyclonal B-cell population under conditions enabling the production of the polyclonal IgG preparation from the polyclonal B-cell population. This improved method enables the production of antibodies (preferably IgG) and facilitates long-term culture of polyclonal B-cell populations.

What is claimed is: 
     
         1 . A human polyclonal IgG preparation prepared by an in vitro method, said method comprising:
 a) obtaining an initial human polyclonal B-cell population;   b) prior to culture, substantially depleting IgM-secreting cells and IgD-secreting cells from the initial human polyclonal B-cell population so as to obtain a human polyclonal B-cell population enriched in IgG-secreting cells;   c) placing the human polyclonal B-cell population enriched in IgG secreting cells in a culture medium; and   d) culturing the human polyclonal B cell population enriched in IgG-secreting cells under conditions enabling the production of the human polyclonal IgG preparation from the polyclonal B cell population;   
       wherein the human polyclonal B cell population of step b) has a concentration of IgM-secreting cells of less than 5% and a concentration of IgD-secreting cells of less than 5%. 
     
     
         2 . The human polyclonal IgG preparation of  claim 1 , wherein the human polyclonal B-cell population of step b) has a concentration of IgG-secreting cells of more than 40%. 
     
     
         3 . The human polyclonal IgG preparation of  claim 1 , wherein the human polyclonal B-cell population of step b) further comprises IgA-secreting cells. 
     
     
         4 . The human polyclonal IgG preparation of  claim 1 , wherein the human polyclonal B-cell population of step b) further comprises IgE-secreting cells. 
     
     
         5 . The human polyclonal IgG preparation of  claim 1 , wherein the human polyclonal B-cell population is derived from peripheral blood. 
     
     
         6 . The human polyclonal IgG preparation of  claim 1  further comprising at least one of the following gamma isotypes of IgG antibodies: IgG 1 , IgG 2 , IgG 3  and IgG 4 . 
     
     
         7 . The human polyclonal IgG preparation of  claim 1  further comprising all the following gamma isotypes of IgG antibodies: IgG 1 , IgG 2 , IgG 3  and IgG 4 . 
     
     
         8 . The human polyclonal IgG preparation of  claim 1 , wherein the relative proportions of IgG isotypes in the human polyclonal IgG preparation are similar to the relative proportions of IgG isotypes in human blood. 
     
     
         9 . The human polyclonal IgG preparation of  claim 1  having a concentration of IgG of more than  70 % when compared to the total Ig content of the human polyclonal IgG preparation. 
     
     
         10 . The human polyclonal IgG preparation of  claim 1  having a concentration of IgM of less than 10% when compared to the total Ig content of the human polyclonal IgG preparation. 
     
     
         11 . The human polyclonal IgG preparation of  claim 1 , wherein the concentration of the human polyclonal IgG preparation in the culture medium supernatant is more than 10 μg/mL. 
     
     
         12 . The human polyclonal IgG preparation of  claim 1 , wherein the human polyclonal B-cell population has been cultured for more than 30 days. 
     
     
         13 . The human polyclonal IgG preparation of  claim 1 , wherein the IgM secretion rate in the human polyclonal IgG preparation is equal to or less than 1000 ng per 10 6  B cells per day. 
     
     
         14 . The human polyclonal IgG preparation of  claim 1 , wherein said method further comprises harvesting the human polyclonal IgG preparation from the culture medium supernatant. 
     
     
         15 . The human polyclonal IgG preparation of  claim 1 , wherein the culture medium comprises at least one of the following: interleukin(IL)-2, IL-4 and IL-10. 
     
     
         16 . A human polyclonal B-cell population prepared by an in vitro method, said method comprising:
 a) substantially depleting IgM-secreting cells and IgD-secreting cells from an initial human polyclonal B cell population prior to culture to provide a human polyclonal B-cell population enriched in IgG-secreting cells;   b) placing the human polyclonal B-cell population enriched in IgG-secreting cells in a culture medium; and   c) culturing the human polyclonal B-cell population enriched in IgG-secreting cells under conditions enabling the multiplication of the polyclonal B-cell population so as to obtain the human polyclonal B-cell population;   
       wherein the human polyclonal B-cell population enriched in IgG-secreting cells of step b) has a concentration of IgM-secreting cells of less than 5% and a concentration of IgD-secreting cells of less than 5%. 
     
     
         17 . The human polyclonal B-cell population of  claim 16 , wherein the human polyclonal B-cell population of step b) has a concentration of IgG-secreting cells of more than 40%. 
     
     
         18 . The human polyclonal B-cell population of  claim 16  further comprising IgA-secreting cells. 
     
     
         19 . The human polyclonal B-cell population of  claim 16  further comprising IgE-secreting cells. 
     
     
         20 . The human polyclonal B-cell population of  claim 16  being derived from peripheral blood. 
     
     
         21 . The human polyclonal B-cell population of  claim 16 , wherein the relative proportions of IgG isotype-secreting cells in the human polyclonal B-cell population are similar to the relative proportions of IgG isotype-secreting B cells in human blood. 
     
     
         22 . The human polyclonal B-cell population of  claim 16 , wherein, in step c) the human polyclonal B-cell population has been cultured for more than 30 days. 
     
     
         23 . The human polyclonal B-cell population of  claim 16 , wherein the culture medium comprises at least one of the following: interleukin(IL)-2, IL-4 and IL-10.