Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule

Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation, whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future mechanistic studies of oxidative degradation of protein therapeutics

Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS following Incubation at Elevated Temperatures

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marke

Specific ion chromatograms (SICs) of Mab1 peptides containing LC-Asx-56.

<p>M = 841.47 Da <i>(z = 1, and 2)</i> (LC-Asp-56; LC-iso-Asp-56) and M = 823.46 Da <i>(z = 1, and 2)</i> (LC-Asu-56) from the Mab1 reference material and a stressed sample (stored at 40°C for 2 month).</p

Analysis of Mab1 target binding by surface plasmon resonance.

<p>Biacore sensorgrams showing the target binding of non-stressed Mab1 material (stored at −80°C) and following elevated temperatures (storage at 40°C for 7 days, 1 month, and 2 month) at a flow rate of 100 µl/min.</p

Low-energy CID mass spectra and resulting amino acid sequence of the doubly protonated Mab1 light chain peptide containing Asp-56 (upper panel) and Asu-56 (lower panel) at <i>m/z</i> 421.7 and 412.7.

<p>Low-energy CID mass spectra and resulting amino acid sequence of the doubly protonated Mab1 light chain peptide containing Asp-56 (upper panel) and Asu-56 (lower panel) at <i>m/z</i> 421.7 and 412.7.</p

ESI-QTOF mass spectrometry of reduced HER2.

<p>NanoESI-QTOF mass spectra of HER2 reference material and HER2 stressed sample (stored at 40°C for 2 month). The spectra were recorded in the positive ion mode using acetonitrile/water/formic acid (78/20/2, v/v/v) as solvent. LC, light chain; HC-G0, non-galactosylated heavy chain; HC-G1, mono-galactosylated heavy chain.</p

Overview of sample preparation procedures A and B for the UPLC-MS analysis of antibody Asn deamidation and Asp isomerization.

<p>Overview of sample preparation procedures A and B for the UPLC-MS analysis of antibody Asn deamidation and Asp isomerization.</p

Specific ion chromatograms (SICs) of Mab1 peptides containing LC-Asx-99/101.

<p>M = 5464.46 Da <i>(z = 3, 4, and 5)</i> (HC-Asp-99/101; HC-iso-Asp-99/101) and M = 5446.45 Da <i>(z = 3, 4, and 5)</i> (HC-Asu-99/101) from the Mab1 reference material and a stressed sample (stored at 40°C for 2 month).</p