title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II).
abstract: BACKGROUND
  The ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development. For example, antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology, where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression, the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE), which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation.
  
  
  RESULTS
  The yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen, driving enzymatic turnover. Building upon that work, here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE, which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture, as demonstrated in fed-batch cell cultures with antibody titers of 5 g · L(-1), specific cellular production rates of 75 pg · cell(-1) · d(-1), and fGly conversion yields of 95-98 %.
  
  
  CONCLUSIONS
  We describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE, which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate.
authors.affiliation: Catalent Pharma Solutions, 726 Heartland Trail, Madison, WI, 53717, USA. dona.york@catalent.com.
authors.forename: Dona
authors.initials: D
authors.lastname: York
authors.affiliation: Catalent Pharma Solutions, 5703 Hollis Street, Emeryville, CA, 94608, USA. jeannebaker2010@gmail.com.
authors.forename: Jeanne
authors.initials: J
authors.lastname: Baker
authors.affiliation: Catalent Pharma Solutions, 5703 Hollis Street, Emeryville, CA, 94608, USA. pgholder@gmail.com.
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Holder
authors.affiliation: Catalent Pharma Solutions, 5703 Hollis Street, Emeryville, CA, 94608, USA. jones.lesleyc@gmail.com.
authors.forename: Lesley C
authors.initials: LC
authors.lastname: Jones
authors.affiliation: Catalent Pharma Solutions, 5703 Hollis Street, Emeryville, CA, 94608, USA. penelope.drake@catalent.com.
authors.forename: Penelope M
authors.initials: PM
authors.lastname: Drake
authors.affiliation: Catalent Pharma Solutions, 5703 Hollis Street, Emeryville, CA, 94608, USA. robyn.barfield@catalent.com.
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Barfield
authors.affiliation: Catalent Pharma Solutions, 726 Heartland Trail, Madison, WI, 53717, USA. gregory.bleck@catalent.com.
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Bleck
authors.affiliation: Catalent Pharma Solutions, 5703 Hollis Street, Emeryville, CA, 94608, USA. david.rabuka@catalent.com.
authors.forename: David
authors.initials: D
authors.lastname: Rabuka
mesh_terms: D000447:Aldehydes / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D003300:Copper / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry*; D005998:Glycine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D000906:Antibodies; D003470:Culture Media; D003300:Copper; D005998:Glycine