Pertuzumab variants and evaluation thereof

Abstract

The present application discloses variants of Pertuzumab. In particular, it discloses: an unpaired cysteine variant comprising Cys23/Cys88 unpaired cysteines in one or both variable light domains of Pertuzumab, an afucosylated variant of Pertuzumab, a low-molecular-weight-species (LMWS) of Pertuzumab, and a high-molecular-weight-species (HMWS) or Pertuzumab. The application further discloses the isolated variants, compositions, pharmaceutical compositions, and articles of manufacture comprising the variants, as well as methods of making and characterizing the variants and compositions thereof

What is claimed is: 
     
         1 . A method for evaluating a Pertuzumab composition comprising measuring the amount of low-molecular-weight-species (LMWS) and high-molecular-weight-species (HMWS) of Pertuzumab in the composition comprising measuring the amount of LMWS and HMWS by size exclusion high performance liquid chromatography (SE-HPLC) wherein the amount of LMWS in the composition is ≤about 1.6% and the amount of HMWS in the composition is ≤about 1.7% as measured by SE-HPLC. 
     
     
         2 . The method of  claim 1 , wherein the amount of LMWS is ≤about 1.2% and the amount of HMWS is ≤about 1.4% as measured by SE-HPLC. 
     
     
         3 . A method for making a composition comprising: (1) producing a composition comprising Pertuzumab and one or more variants thereof, and (2) subjecting the composition so-produced to an analytical assay to evaluate the amount of the variant(s) therein, wherein the variant(s) comprise: high-molecular-weight-species (HMWS) and low-molecular-weight-species (LMWS) of Pertuzumab, wherein the analytical assay measures the amount of LMWS and HMWS by size exclusion high performance liquid chromatography (SE-HPLC) and the amount of LMWS in the composition is ≤about 1.6% and the amount of HMWS in the composition is ≤about 1.7% as measured by SE-HPLC. 
     
     
         4 . The method of  claim 3 , wherein (1) comprises expressing Pertuzumab and the variant(s) from a recombinant Chinese Hamster Ovary (CHO) cell at manufacturing scale and purifying the composition. 
     
     
         5 . The method of  claim 4 , wherein the amount of LMWS is ≤about 0.6% and the amount of HMWS is ≤about 0.8% as measured by SE-HPLC. 
     
     
         6 . The method of  claim 4 , wherein the amount of LMWS is ≤about 1.2% and the amount of HMWS is ≤about 1.4% as measured by SE-HPLC. 
     
     
         7 . The method of  claim 3 , wherein the amount of LMWS is ≤about 0.6% and the amount of HMWS is ≤about 0.8% as measured by SE-HPLC.