Host cell protein (HCP) impurities present at low ppm levels in protein biopharmaceuticals are challenging to detect and more challenging to identify within the encompassing biotherapeutic matrix. Multiple separation dimensions in an HCP LCMS discovery workflow are clearly beneficial to achieve the chromatographic resolution of these low level impurities, while enabling the high sample loading to overcome the dynamic range inherent to this complex analysis. Our laboratory developed and previously published a novel 2D (High pH/Low pH Reversed Phase) peptide digest separation coupled to MSE based data independent MS/MS detection approach for HCP identification down to sub-50 ppm levels [Doneanu et. al. mAbs, 2012, 4, 24-44.]. This poster details three improvements to this methodology required to nominally achieve 1 ppm (1 ng HCP/mg mAb) HCP identification levels from purified mAb samples: Use of a Charged Surface Hybrid (CSH) UPLC chemistry enabled 5-fold higher peptide loading, to raise more peptides above assay detection limits; the use of ion mobility MS based HDMSE acquisition workflows provided additional peptide resolution, for clean detection of lower level components; and, the use of optimized fragmentation energy profiles based on peptide ion mobility drift profile improved fragment ion spectra, for superior HCP peptide identification. The robustness of this second generation HCP discovery methodology was demonstrated by a cross-laboratory (n=3) study using the new NIST mAb standard as a common sample for the analysis. This approach was also used to identify HCP profiles from innovator vs. biosimilar Infliximab products. These results demonstrate that LC/MS assays are able to attain comparable sensitivity to traditional HCP assays (e.g. ELISA), while offering unique advantages of individual HCP identification and quantification.