Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p

Inflammatory profiles of <i>B</i>. <i>burgdorferi</i> ss, parental strain and its clone 297/4.

<p>Levels of transcripts were measured by RT-qPCR from the skin at the inoculation site. The values were calculated using the 2-delta delta Ct method after normalization with <i>gapdh</i>. (h: hours, d: days). Two-way ANOVA was used to analyze the data. At least, three mice were analyzed for each time point.</p

Proteomic analyses of <i>B</i>. <i>burgdorferi</i> ss, 297 parental strain and its clone 297/4.

<p>(A) The Venn diagram shows the protein overlap and the proteins specifically identified in the parental strain and the clone 297/4. (B) Graphic representation of the breakdown of the proteins specifically identified in the clone. Categorization was based upon JCVI annotation. The percentages represent the fraction of that category within the proteins. (C) Gene location of the specific proteins identified in the clone 297/4.</p

Bacterial loads in the skin.

<p>The spirochetal burden in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>. Values represent relative expression + SD of three independent experiments. Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). (d: days).</p

Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p

Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment

Antibody drug conjugates (ADCs) are macromolecules composed of cytotoxic drugs covalently attached via a conditionally stable linker to monoclonal antibodies (mAbs). ADCs are among the most promising next generation of empowered mAbs foreseen to treat cancers. Compared to naked mAbs, ADCs have an increased level of complexity as the heterogeneity of conjugation cumulates with the inherent microvariability of the biomolecule. An increasing need underlying ADC’s development and optimization is to improve its analytical and bioanalytical characterization by assessing three main ADC quality attributes: drug distribution, amount of naked antibody, and average drug to antibody ratio (DAR). Here, the analytical potential of native mass spectrometry (MS) and native ion mobility MS (IM-MS) is compared to hydrophobic interaction chromatography (HIC), the reference method for quality control of interchain cysteinyl-linked ADCs. Brentuximab vedotin, first in class and gold standard, was chosen for a proof of principle. High resolution native MS provided accurate mass measurement (<30 ppm) of intact ADCs together with average DAR and drug distribution, confirming the unique ability of native MS for simultaneous detection of mixtures of covalent and noncovalent products. Native IM-MS was next used for the first time to characterize an ADC. IM-MS evidenced ADC multiple drug loading, collisional cross sections measurement of each payload species attesting slight conformational changes. A semiquantitative interpretation of IM-MS data was developed to directly extrapolate average DAR and DAR distribution. Additionally, HIC fractions were collected and analyzed by native MS and IM-MS, assessing the interpretation of each HIC peak. Altogether, our results illustrate how native MS and IM-MS can rapidly assess ADC structural heterogeneity and how easily these methods can be implemented into MS workflows for in-depth ADC analytical characterization

Inflammatory profiles of the different strains of <i>B</i>. <i>burgdorferi</i> ss.

<p>Levels of transcripts were measured by RT-qPCR from the skin at the inoculation site. The values were calculated using the 2-delta delta Ct method after normalization with <i>gapdh</i>. (h: hours, d: days). Two-way ANOVA was used to analyze the data. At least three mice were analyzed for each time point.</p