Glycan structures attached to the C(H)2 domain of the Fc region of immunoglobulin G (IgG) are essential for specific effector functions but their role in modulating clearance is less clear. Clearance is of obvious importance for therapeutic monoclonal antibodies (Mabs) as it directly impacts efficacy. Here, we study the impact of Fc glycan structure on the clearance of four therapeutic human IgGs (one IgG1 and three IgG2s) in humans. The therapeutic IgGs were affinity purified from serum samples from human pharmacokinetic studies, and changes to the glycan profile over time were determined by peptide mapping employing high-resolution mass spectrometry. Relative levels of high-mannose 5 (M5) glycan decreased as a function of circulation time, whereas other glycans remained constant. These results demonstrate that therapeutic IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other glycan forms. The quantitative effect of this on pharmacokinetic area under the curve was calculated and shown to be relatively minor for three of the four molecules studied, but, depending on the dosing regimen and the relative level of the high-mannose glycan, this can also have significant impact. High-mannose content of therapeutic Mabs should be considered an important product quality attribute which may affect pharmacokinetic properties of therapeutic antibodies.

The conformations of the NeuAcα2(I)→3Galβ1(II)→4[Fucα1(III)→3]GlcNAc-O-CH3 tetrasaccharide (sLex), in aqueous solution and bound to E-, P-, and L-selectin have been determined using high resolution NMR spectroscopy. In the free ligand, the conformation of glycosidic linkage I is disordered with {ΦI, ΨI} sampling values close to {−60°, 0°}, {−100°, −50°}, and {180°, 0°}. The trisaccharide portion is rigid and characterized by {ΦII, ΨII; ΦIII, ΨIII} = {46°, 18°; 48°, 24°}. The measured dissociation rates and equilibrium binding constants, {k off, K D}, were {164 ± 24 s-1, 0.72 ± 0.4 mM}, {522 ± 166 s-1, 7.8 ± 1.0 mM}, and {1080 ± 167 s-1, 3.9 ± 0.6 mM} at 300 K for E-, P-, and L-selectin, respectively. The bound conformations of the ligand were calculated from the full relaxation matrix analysis of transferred-NOE spectra for E- and P-selectin or by using a two-spin approximation for the L-selectin complex. Both E- and P-selectin recognize the {−60°, 0°} conformation of sLex while the {−100°, −50°} conformer is probably recognized by L-selectin. The conformation of the branched trisaccharide portion in the bound state remains close to the conformation of the free ligand. In the E-, P-, and L-selectin complexes the GalH4 proton is in the vicinity of protein aromatic protons, most likely Tyr94 and/or Tyr48.

A mass spectrometry-based method was developed to measure amino acid substitutions directly in proteins down to a level of 0.001%. When applied to recombinant proteins expressed in Escherichia coli, monoclonal antibodies expressed in mammalian cells, and human serum albumin purified from three human subjects, the method revealed a large number of amino acid misincorporations at levels of 0.001-0.1%. The detected misincorporations were not random but involved a single-base difference between the codons of the corresponding amino acids. The most frequent base differences included a change from G to A, corresponding to a G(mRNA)/U(tRNA) base pair mismatch during translation. We concluded that under balanced nutrients, G(mRNA)/U(tRNA) mismatches at any of the three codon positions and certain additional wobble position mismatches (C/U and/or U/U) are the main causes of amino acid misincorporations. The hypothesis was tested experimentally by monitoring the levels of misincorporation at several amino acid sites encoded by different codons, when a protein with the same amino acid sequence was expressed in E. coli using 13 different DNA sequences. The observed levels of misincorporation were different for different codons and agreed with the predicted levels. Other less frequent misincorporations may occur due to G(DNA)/U(mRNA) mismatch during transcription, mRNA editing, U(mRNA)/G(tRNA) mismatch during translation, and tRNA mischarging.

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A technique for rapid characterization of variable regions of monoclonal antibodies (mAb) is described. Several intact mAbs were analyzed on a Thermo-Fisher LTQ-Orbitrap high-resolution mass spectrometer (MS) by in-source fragmentation. In-source fragmentation has the unique advantage of fragmenting all charge states of a protein at the same time and, thus, greatly improves the sensitivity of the fragment ions over a true MS/MS experiment, where a single charge state is isolated and fragmented. In addition, immediate fragmentation of the protein before tertiary structure formation may also facilitate protein fragmentation. This technique has been proved very useful for top-down analysis of large proteins. In-source fragmentation of mAbs generated a series of fragment ions. In addition to some small b and y ions from the light chain and heavy chain in the low m/z region, a series of b ions corresponding to N-terminal 106-120 residues of both heavy chain and light chain were observed. The cleavage sites for these b ions happen to be near the linker regions between the variable domains and the constant domains of these antibodies. These b ions, therefore, correspond to the entire variable region of each chain. Similar results were obtained for all mAbs analyzed, including both immunoglobulin G1 and G2 molecules. To further characterize the variable regions, these b ions were isolated and fragmented by collision-induced dissociation in the linear trap, followed by mass analysis in the orbitrap. Large number of product ions was observed from these b ions. Many of these product ions are internal fragments between the two disulfide-linked cysteine residues. To demonstrate the capability of the technique, several mAbs were force-oxidized by treating with tert-butyl hydroperoxide, followed by mass spectrometric analysis. In-source fragmentation and MS/MS of the variable region b ions clearly identified the locations of the oxidized methionine.