System and method for characterizing drug product impurities

Abstract

Systems and methods for characterizing low molecular weight (LMW) protein drug product impurities are provided. One embodiment uses hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry analysis. After removal of the N-linked glycans from the protein drug product, for example an antibody drug product, the elution of LMW impurities from the HILIC column was determined by the size of the molecular weight species. In some embodiments, the HILIC separation is performed under denaturing conditions, making the detection of LMW forms using this method highly comparable to both SDS-PAGE and CE-SDS methods. LMW drug product impurities include, but are not limited to light chain, half antibody, H2L, H2, HL, HC, peptide backbone-truncated species, and combinations thereof.

We claim: 
     
         1 . A protein drug product comprising:
 a protein drug and an excipient, wherein the protein drug product comprises between 0.05% and 30.0% (w/w) of low molecular weight protein drug impurities.   
     
     
         2 . The protein drug product of  claim 1 , wherein the protein drug product is selected from the group consisting of an antibody, a fusion protein, recombinant protein, or a combination thereof. 
     
     
         3 . The protein drug product of  claim 1 , wherein the low molecular weight protein drug impurities are selected from the group consisting of degradation products, free light chain, half antibody, H2L, H2, HL, HC, or a combination thereof. 
     
     
         4 . The protein drug product of  claim 1 , wherein the drug product comprises between 0.05% to 25% w/w of low molecular weight protein drug impurities. 
     
     
         5 . The protein drug product of  claim 1 , wherein the drug product comprises between 0.05% to 15% w/w of low molecular weight protein drug impurities. 
     
     
         6 . The protein drug product of  claim 1 , wherein the drug product comprises between 0.05% to 10% w/w of low molecular weight protein drug impurities. 
     
     
         7 . The protein drug product of  claim 1 , wherein the drug product comprises between 0.05% to 5% w/w of low molecular weight protein drug impurities. 
     
     
         8 . A method for characterizing low molecular weight protein drug product impurities comprising:
 i) deglycosylating a protein drug product sample;   ii) separating protein components of the protein drug product sample by hydrophilic interaction chromatography;   iii) analyzing the separated protein components by mass spectroscopy to characterize low molecular weight protein drug product impurities in the protein drug product sample, wherein the protein drug product is as defined in  claim 1 .   
     
     
         9 . The method of  claim 8 , wherein the protein drug product sample is from a fed-batch culture. 
     
     
         10 . The method of  claim 8 , wherein the protein drug product is selected from the group consisting of an antibody, a fusion protein, recombinant protein, or a combination thereof. 
     
     
         11 . The method of  claim 8 , wherein the low molecular weight protein drug product impurity is characterized as a low molecular weight protein drug product impurity selected from the group consisting of free light chain, half antibody, H2L, H2, HL, HC, or a combination thereof. 
     
     
         12 . A method of producing an antibody, comprising:
 i) culturing cells producing the antibody in a cell culture;   ii) obtaining a sample from the cell culture;   iii) characterizing and quantifying low molecular weight impurities in the sample according to the method of  claim 8 , and   iv) modifying one or more culture conditions of the cell culture to reduce the amount of characterized low molecular protein drug impurities produced during cell culture of the antibody.   
     
     
         13 . The method of  claim 12 , wherein the one or more conditions of the cell culture that are changed to reduce the amount of low molecular weight protein drug impurities are selected from the group consisting of pH, cell density, amino acid concentration, osmolality, growth factor concentration, agitation, gas partial pressure, surfactants, or combinations thereof. 
     
     
         14 . The method of  claim 12 , wherein the cells are selected from the group consisting of bacterial cells, yeast cells, Chinese Hamster Ovary (CHO) cells (e.g. CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g. COS-7), retinal cells, Vero cells, CV1 cells, kidney cells (e.g. HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo25 cells, HB 8065 cells, HL-60 cells, lymphocyte cells, e.g. autologous T cells, Jurkat (T lymphocytes) or Daudi (B lymphocytes), A431 (epidermal) cells, U937 cells, 3T3 cells, L cells, C127 cells, SP2/0 cells, NS-0 cells, MMT cells, stem cells, tumor cells, and a cell line derived from any of the aforementioned cells. 
     
     
         15 . The method of  claim 12 , wherein the cells are hybridoma cells or quadroma cells. 
     
     
         16 . An antibody produced by the method of  claim 12 . 
     
     
         17 . The antibody of  claim 16 , comprising 0.05 and 30.0% (w/w) of low molecular weight protein drug impurities. 
     
     
         18 . A system for characterizing low molecular weight drug impurities, comprising:
 a hydrophilic interaction liquid chromatography system comprising a hydrophilic interaction liquid chromatography (HILIC) column linked to at least two mobile phase columns, wherein the HILIC column is in fluid communication with a mass spectroscopy system.   
     
     
         19 . A method for determining the stability and forced degradation studies of a protein drug product using the system of  claim 18 .