No abstract

Misincorporation of amino acids in proteins expressed inEscherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA Asn rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions. Many recombinant proteins have been approved as therapeutic drugs by the Food and Drug Administration, and many more are undergoing clinical trial (1). For economic and practical reasons, considerable effort has been made to increase product yield and process efficiency for proteins made in mammalian cell culture. Nowadays, large amounts of proteins can be expressed efficiently in optimized expression systems, with yields from bioreactors having improved more than 100-fold during the past two decades (2). Yields as high as 10 g/liter have been reported for production of monoclonal antibodies in CHO 2 cells (3). These yields are due mainly to improvements in host cell engineering, cell line selection, and culture medium optimization (4). However, it is well known that overexpressing recombinant proteins can lead to nutritional stresses in the host cells and that these stresses can markedly increase the frequency of random translational errors, resulting in a heterogeneous mixture of proteins (5-11). A variety of translational errors have been observed during overexpression of proteins in Escherichia coli, including frame shifts, premature truncation, read-through, leaky stop codons, and amino acid misincorporation (12-16). Nevertheless, there are few such reports for proteins made in mammalian cells, and it is commonly believed that the fidelity of translation in mammalian cells is higher (8, 17). Here we report for the first time that misincorporation, namely of Ser for Asn, can occur in proteins overexpressed in CHO cells under normal recombinant protein production conditions. Further investigation showed that supplementation of the medium with Asn can overcome this problem. Our work demonstrates that protein products should be closely monitored for misincorporation, for example, by molecular mass determination and peptide mapping during optimization of culture conditions. EX...

Despite the great success of mass spectrometry (MS) for de novo protein sequencing, Leu and Ile have been generally considered to be indistinguishable by MS because their molecular masses are exactly the same. Positioning of incorrect Leu/Ile residues in variable domains, especially in CDRs (complementarity determining regions) of an antibody, may result in substantial loss of antigen binding affinity and specificity of the antibody. Here, we describe an integrated LC-MS based strategy, encompassing a combination of HCD (high-energy collisional dissociation) multistage mass spectrometric analysis (HCD-MS) and ETD (electron transfer dissociation)-HCD MS analysis using an Orbitrap Fusion mass spectrometer, to reliably identify Leu and Ile residues in proteins and peptides. The merits and limitations of this Leu/Ile discrimination approach are evaluated. Using the new approach, along with proposed decision-making guidelines we unambiguously identified every Leu/Ile residue in peptides containing up to five Leu/Ile residues and molecular masses up to 3000 Da. In addition, we have demonstrated, for the first time, that every Leu/Ile residue in the variable regions of a monoclonal antibody that could not be assigned by antibody germline sequence alignment could be correctly determined using this approach. Our results suggest that, by incorporating this approach into existing de novo antibody sequencing protocols, 100% of antibody amino acid sequences, including identity of Leu and Ile residues, can be accurately obtained solely by means of mass spectrometry. In principle, this integrated, online LC-MS approach for Leu/Ile assignment can be applied to de novo sequencing of any protein or peptide.

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.

Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains – two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the Cϵ2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDB ID 6CR1).