Process for producing exogenous protein in the milk of transgenic mammals and a process for purifying proteins therefrom

Abstract

The invention relates to a method of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct comprising the hGH gene and beta casein promoter sequences conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

1 .- 29 . (canceled) 
     
     
         30 . A method of purifying a recombinant growth hormone from milk of a non-human transgenic mammal of bovine species that produces a recombinant growth hormone in its milk, wherein said mammal produces increased growth hormone in its serum over a nontransgenic mammal of bovine species comprising:
 a) clarifying the milk of said non-human transgenic mammal, resulting in a clarified milk; and   b) subjecting said clarified milk to chromatography, resulting in purified recombinant growth hormone.   
     
     
         31 . The method of  claim 30 , wherein said chromatography is ion exchange chromatography, reverse phase chromatography, molecular exclusion chromatography or affinity chromatography. 
     
     
         32 . The method of  claim 31 , wherein multiple chromatography steps are performed. 
     
     
         33 . The method of  claim 31 , wherein said ion exchange chromatography is anion exchange chromatography. 
     
     
         34 . The method of  claim 31 , wherein the affinity chromatography is immunochromatography. 
     
     
         35 . The method of  claim 31 , further comprising a concentration step. 
     
     
         36 . (canceled) 
     
     
         37 . A method of purifying a recombinant growth hormone from milk of a non-human transgenic mammal of bovine species that produces a recombinant growth hormone in its milk, wherein said mammal produces increased growth hormone in its serum over a nontransgenic mammal of bovine species comprising:
 a) clarifying milk obtained from a transgenic mammal, resulting clarified milk;   b) subjecting said clarified milk to immunoaffinity chromatography, resulting in an immunoaffinity chromatographed material;   c) subjecting said immunoaffinity chromatographed material reverse phase chromatography, resulting in a reverse phase chromatographed material;   d) subjecting said reverse phase chromatographed material to anionic exchange chromatography, resulting in an anionic exchange chromatographed material;   e) subjecting said anionic exchange chromatographed material to molecular exclusion chromatography, resulting in a molecular exclusion chromatographed material;   f) subjecting said molecular exclusion chromatographed material to concentration, resulting in a concentrated material; and   g) subjecting said concentrated material to molecular exclusion chromatography, resulting in pure recombinant growth hormone.   
     
     
         38 . The method of  claim 37 , wherein said growth hormone is a mammalian growth hormone. 
     
     
         39 . The method of  claim 38 , wherein said mammalian growth hormone is human growth hormone, bovine growth hormone, porcine growth hormone, ovine growth hormone, caprine growth hormone or rodent growth hormone. 
     
     
         40 . The method of  claim 39 , wherein said mammalian growth hormone is human growth hormone. 
     
     
         41 . The method of  claim 40 , wherein said mammal produces human growth hormone at a level of at least about 1.0 g hGH/L milk. 
     
     
         42 . The method of  claim 41 , wherein said mammal produces human growth hormone at a level of at least about 2.0 g hGH/L milk. 
     
     
         43 . The method of  claim 42 , wherein said mammal produces human growth hormone at a level of at least about 3.0 g hGH/L milk. 
     
     
         44 . The method of  claim 43 , wherein said mammal produces human growth hormone at a level of at least about 4.0 g hGH/L milk. 
     
     
         45 . The method of  claim 44 , wherein said mammal produces human growth hormone at a level of at least about 5.0 g hGH/L milk. 
     
     
         46 . The method of  claim 45 , wherein said mammal produces human growth hormone at a level of at least about 6.0 g hGH/L milk. 
     
     
         47 .- 48 . (canceled)