Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality.

[1]  Qin Fu,et al.  Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. , 2009, Current protocols in molecular biology.

[2]  Pornpimol Charoentong,et al.  ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks , 2009, Bioinform..

[3]  D. Lamont,et al.  Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules* , 2005, Journal of Biological Chemistry.

[4]  M. Butler,et al.  Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level , 2000, Cytotechnology.

[5]  Gyun Min Lee,et al.  Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity. , 2016, Biotechnology journal.

[6]  Peter G. Slade,et al.  Identifying the CHO secretome using mucin-type O-linked glycosylation and click-chemistry. , 2012, Journal of proteome research.

[7]  A. Cuervo,et al.  Cathepsin A regulates chaperone‐mediated autophagy through cleavage of the lysosomal receptor , 2003, The EMBO journal.

[8]  B. Kerwin.  Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. , 2008, Journal of pharmaceutical sciences.

[9]  B. Sharma,et al.  Tryptic map variation of erythropoietin resulting from carboxypeptidase B-like activity , 1994 .

[10]  Katherine D Taylor,et al.  Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles. , 2016, Journal of pharmaceutical sciences.

[11]  Bernhard Mlecnik,et al.  CluePedia Cytoscape plugin: pathway insights using integrated experimental and in silico data , 2013, Bioinform..

[12]  R. Ionescu,et al.  Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. , 2008, Current pharmaceutical biotechnology.

[13]  J. S. Ryu,et al.  Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant chinese hamster ovary cell cultures differs among cell lines. , 2000, Biotechnology and bioengineering.

[14]  Hervé Broly,et al.  Degradation of an Fc‐fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease , 2009, Biotechnology and bioengineering.

[15]  T. Raju,et al.  Terminal sugars of Fc glycans influence antibody effector functions of IgGs. , 2008, Current opinion in immunology.

[16]  Michael J. Lewis,et al.  Proteomic Analysis of Bioreactor Cultures of an Antibody Expressing CHOGSCell Line that Promotes High Productivity , 2013 .

[17]  C. Goochee,et al.  Glycosidase Activities in Chinese Hamster Ovary Cell Lysate and Cell Culture Supernatant , 1993, Biotechnology progress.

[18]  W. Hancock,et al.  Characterization of the proteases involved in the N‐terminal clipping of glucagon‐like‐peptide‐1‐antibody fusion proteins , 2011, Biotechnology progress.

[19]  M. Gottesman,et al.  Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts. , 1990, The Biochemical journal.

[20]  E. Mandine,et al.  Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems , 1988, Biotechnology Letters.

[21]  Thomas Ryll,et al.  Maximizing productivity of CHO cell‐based fed‐batch culture using chemically defined media conditions and typical manufacturing equipment , 2010, Biotechnology progress.

[22]  Feng Li,et al.  Cell culture processes for monoclonal antibody production , 2010, mAbs.

[23]  Y. Schneider,et al.  Recombinant interferon-γ secreted by chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium , 2005, In Vitro Cellular & Developmental Biology - Animal.

[24]  P. E. Van den Steen,et al.  Gelatinase B/matrix metalloproteinase‐9 cleaves interferon‐β and is a target for immunotherapy , 2003 .

[25]  Marcel Leist,et al.  Cathepsin B Acts as a Dominant Execution Protease in Tumor Cell Apoptosis Induced by Tumor Necrosis Factor , 2001, The Journal of cell biology.

[26]  B. Alberts,et al.  Transport from the Trans Golgi Network to Lysosomes , 2002 .

[27]  D. Lütkemeyer,et al.  Mapping and partial characterization of proteases expressed by a CHO production cell line , 2006, Biotechnology and bioengineering.

[28]  John F. Valliere-Douglass,et al.  Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non‐denaturing IEF fractionation , 2015, Electrophoresis.

[29]  P. Briza,et al.  Structure and Mechanism of an Aspartimide-Dependent Peptide Ligase in Human Legumain** , 2015, Angewandte Chemie.

[30]  J. F. Nemeth,et al.  Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide‐1‐MIMETIBODYTM: Factors that influence productivity and product quality , 2009, Biotechnology and bioengineering.

[31]  Tilman Schlothauer,et al.  In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity , 2015, PloS one.

[32]  Pamela Stanley,et al.  Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs) , 2001, The Journal of Biological Chemistry.

[33]  A. Goldberg,et al.  Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes* , 2012, The Journal of Biological Chemistry.

[34]  C. Barrow,et al.  Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization , 2016, International journal of molecular sciences.

[35]  Lihua Huang,et al.  Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A2 Isomer X1 in Monoclonal Antibody Formulations. , 2016, Journal of pharmaceutical sciences.

[36]  C. Smales,et al.  Host cell protein dynamics in the supernatant of a mAb producing CHO cell line , 2012, Biotechnology and bioengineering.

[37]  Wei Wang,et al.  Antibody structure, instability, and formulation. , 2007, Journal of pharmaceutical sciences.

[38]  C. Goochee,et al.  Glycosidase activities of the 293 and NS0 cell lines, and of an antibody‐producing hybridoma cell line , 1994, Biotechnology and bioengineering.

[39]  B. Ferrari,et al.  Comparative Protein Expression in Different Strains of the Bloom-forming Cyanobacterium Microcystis aeruginosa* , 2011, Molecular & Cellular Proteomics.

[40]  Renate Kunert,et al.  Benchmarking of commercially available CHO cell culture media for antibody production , 2015, Applied Microbiology and Biotechnology.

[41]  Shujun Bai,et al.  Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity. , 2011, Biotechnology and bioengineering.

[42]  Sandeep Yadav,et al.  Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism. , 2015, Molecular pharmaceutics.

[43]  Chew-Kiat Heng,et al.  Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. , 2005 .

[44]  Difei Qiu,et al.  C‐terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes , 2008, Biotechnology and bioengineering.

[45]  Martin Dragosits,et al.  Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool , 2013, Applied Microbiology and Biotechnology.

[46]  Liu Tie,et al.  Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product , 2015, Biotechnology progress.

[47]  Edward J. O'Brien,et al.  Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome , 2013, Nature Biotechnology.

[48]  Nathan E Lewis,et al.  Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics. , 2015, Journal of proteome research.

[49]  David Robbins,et al.  A Novel Approach to Monitor Clearance of Host Cell Proteins Associated With Monoclonal Antibodies , 2014, Biotechnology progress.

[50]  Andreas Rizzi,et al.  Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[51]  J. Chon,et al.  Advances in the production and downstream processing of antibodies. , 2011, New biotechnology.

[52]  D. James,et al.  Control of Recombinant Monoclonal Antibody Effector Functions by Fc N‐Glycan Remodeling in Vitro , 2005, Biotechnology progress.

[53]  Yelena Lyubarskaya,et al.  Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity. , 2006, Analytical biochemistry.