title: Chromatin function modifying elements in an industrial antibody production platform--comparison of UCOE, MAR, STAR and cHS4 elements.
abstract: The isolation of stably transfected cell lines suitable for the manufacture of biotherapeutic protein products can be an arduous process relying on the identification of a high expressing clone; this frequently involves transgene amplification and maintenance of the clones' expression over at least 60 generations. Maintenance of expression, or cell line stability, is highly dependent upon the nature of the genomic environment at the site of transgene integration, where epigenetic mechanisms lead to variable expression and silencing in the vast majority of cases. We have assessed four chromatin function modifying elements (A2UCOE, MAR X_S29, STAR40 and cHS4) for their ability to negate chromatin insertion site position effects and their ability to express and maintain monoclonal antibody expression. Each element was analysed by insertion into different positions within a vector, either flanking or between heavy chain (HC) and light chain (LC) antibody expression cassettes. Our results clearly show that the A2UCOE is the most beneficial element in this system, with stable cell pools and clones increasing antibody yields 6.5-fold and 6.75-fold respectively. Stability analysis demonstrated that the reduction in antibody expression, seen with cells transfected with the control vector over 120 generations, was mitigated in the clones containing A2UCOE-augmented transgenes. Analysis also showed that the A2UCOE reduced the amount of transgene promoter DNA methylation, which contributed to the maintenance of starting levels of expression.
authors: Protein Expression and Purification Group, UCB, Slough, Berkshire, United Kingdom.
authors: Fay
authors: F
authors: Saunders
authors: Protein Expression and Purification Group, UCB, Slough, Berkshire, United Kingdom.
authors: Berni
authors: B
authors: Sweeney
authors: Gene Expression and Therapy Group, King's College School of Medicine, Department of Molecular Genetics, Guy's Hospital, London Bridge, London, United Kingdom.
authors: Michael N
authors: MN
authors: Antoniou
authors: Protein Expression and Purification Group, UCB, Slough, Berkshire, United Kingdom.
authors: Paul
authors: P
authors: Stephens
authors: Protein Expression and Purification Group, UCB, Slough, Berkshire, United Kingdom.
authors: Katharine
authors: K
authors: Cain
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000235:genetics*; D016466:CHO Cells; D002843:Chromatin / Q000235:genetics*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D003412:Cricetulus; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D019076:Transgenes / Q000502:physiology*
chemical_list: D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains