Anti-Rhesus D Recombinant Polyclonal Antibody and Methods of Manufacture

Abstract

The invention relates to a method for manufacturing an anti-RhD recombinant polyclonal antibody composition (anti-RhD rpAb). The method comprises obtaining a collection of cells transfected with a library of anti-RhD antibody expression vectors, wherein each cell in the collection is capable of expressing from a VH and VL comprising nucleic acid segment, one member of the library, which encodes a distinct member of anti-RhD recombinant polyclonal antibody composition and is located at the same site in the genome of individual cells in said collection. The cells are cultured under suitable conditions for expression of the recombinant polyclonal antibody, which is obtained from the cells or culture supernatant. Nucleic acid segments encoding the anti-RhD rpAb are introduced into the cells by transfection with a library of vectors for site-specific integration. The method is suitable for manufacturing anti-RhD rpAb, thereby making available a superior replacement of plasma-derived prophylactic and therapeutic immunoglobulin products.

1 - 53 . (canceled) 
     
     
         54 . A method for generating a polyclonal working cell bank, said method comprising:
 a) providing a collection of cell lines, obtained from host cells which have been individually transfected with an individual member of a library comprised of variable region-encoding nucleic acid segments, and where each individual cell line upon transfection produces a different member of a polyclonal protein,   b) mixing a predefined number of cells expanded from each of said cell lines, and   c) freezing aliquots of the mixture.   
     
     
         55 . The method according to  claim 54 , wherein an aliquot obtained in step c) is thawed and expanded for a number of generations sufficient to produce a total number of cells, which are frozen down in a new series of aliquots (sub-pWCB), with approximately the same number of cells in each aliquot of said sub-pWCB as in said thawed aliquot. 
     
     
         56 . The method according to  claim 54 , wherein said library comprised of variable region-encoding nucleic acid segments is a library comprised of antibody V H - and V L -encoding nucleic acid segments. 
     
     
         57 . The method according to  claim 56 , wherein said library comprised of antibody V H - and V L -encoding nucleic acid segments encodes an anti-RhD recombinant polyclonal antibody. 
     
     
         58 . The method according to  claim 54 , wherein said individual cell lines are selected, prior to the mixing of the cells, such that they have similar proliferation rates or productivity. 
     
     
         59 . The method according to  claim 58 , wherein said individual cell lines are selected for similar productivity by FACS analysis. 
     
     
         60 . The method according to  claim 58 , wherein said individual cell lines are selected for similar productivity or proliferation rates by means of a robot. 
     
     
         61 . The method according to  claim 58 , wherein said selected cell lines have a proliferation rate between 22 and 40 hours or a productivity exceeding 1.5 pg/(cell×day). 
     
     
         62 . The method according to  claim 54 , wherein said individual cell lines are cloned cell lines. 
     
     
         63 . The method according to  claim 54 , wherein each individual cell line produces a full-length antibody with properties that differ from the properties of the antibodies produced by the other members of the polyclonal working cell bank. 
     
     
         64 . The method according to  claim 54 , wherein said individual cell lines are mixed at equal ratios. 
     
     
         65 . The method according to  claim 54 , wherein said individual cell lines are mixed at different ratios. 
     
     
         66 . The method according to  claim 65 , wherein the ratio of one or more individual cell lines producing an antibody which binds a particular antigen is increased compared to the other members of the polyclonal working cell bank. 
     
     
         67 . The method according to  claim 65 , wherein the ratio of one or more individual cell lines characterized by having a slower proliferation rate is increased compared to other members of the polyclonal working cell bank characterized by a faster proliferation rate. 
     
     
         68 . A polyclonal working cell bank comprising a mixture of a predefined number of cells from a collection of individual cell lines, where each individual cell line is obtained from host cells which have been individually transfected with an individual member of a library comprised of variable region-encoding nucleic acid segments, and where each individual cell line produces a different member of a polyclonal protein, and where said pWCB has been frozen down in aliquots. 
     
     
         69 . The polyclonal working cell bank according to  claim 68 , wherein said individual cell lines have similar proliferation rates or productivity. 
     
     
         70 . The polyclonal working cell bank according to  claim 68 , wherein said individual cell lines are cloned cells. 
     
     
         71 . The polyclonal working cell bank according to  claim 68 , wherein said individual cell lines are mixed at different ratios.