Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

Abstract

Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via cochromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines

“…In addition to proteinogenic amino acids, non-proteinogenic amino acids and rare PTMs can also be detected in the protein sequence [ 19 – 21 ]. One of the more common mammalian non-proteinogenic amino acids is hydroxyproline, a known analog of proline that is found in collagen supporting its triple helix structure and stability [ 22 , 23 ].…”

“…Hydroxylation of proline occurs after protein synthesis and is catalyzed by the enzyme prolyl hydroxylase [ 25 ]. Tyshchuk et al [ 21 ] and Spahr et al [ 23 ] reported the presence of hydroxyproline and a corresponding hydroxylation site in recombinant therapeutics expressed using CHO expression systems. E .…”

Modulating tyrosine sulfation of recombinant antibodies in CHO cell culture by host selection and sodium chlorate supplementation

“…Tyrosine sulfation is a common PTM in mammalian cells but rarely seen on recombinant therapeutic antibodies. The two sulfated antibodies reported so far were both modified in the variable region of light chain, [ 15,16 ] rendering a high chance of interference on antigen binding. In addition, there is potential immunogenicity risk stemming from the sulfated tyrosine.…”

“…also identified a sulfated tyrosine on a recombinant antibody and proposed an in silico method to predict tyrosine sulfation at antibody engineering stage. [ 16 ]…”

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry

“…42 Recently, we applied this approach for the characterization of a sulfated mAb and localized sulfation on a tyrosine of the Lc. 44 Here, we applied a middle-up MS strategy to analyze all six different subunits generated after IdeS digestion and chemical reduction of Ang-2-VEGF (A2V) BsAb (i.e., two different Lc, two different Fd and two different Fc/2) in a single mass spectrum. This allowed monitoring of glycation levels during a forced-glycation experiment performed on intact A2V BsAb.…”