Multiple reaction monitoring (MRM) is a liquid chromatography−mass spectrometry (LC−MS) based quantification platform with high sensitivity, specificity, and throughput. It is extensively used across the pharmaceutical industry for the quantitative analysis of therapeutic molecules. The potential of MRM analysis for the quantification of specific host cell proteins (HCPs) in bioprocess, however, has yet to be well established. In this work, we introduce a multiplex LC−MRM assay that simultaneously monitors two high risk lipases known to impact biologics product quality, Phospholipase B-like 2 protein (PLBL2) and Group XV lysosomal phospholipase A2 (LPLA2). Quantitative data generated from the LC−MRM assay were used to monitor the clearance of these lipases during biologics process development. The method is linear over a dynamic range of 1 to 500 ng/ mg. To demonstrate the fitness for use and robustness of this assay, we evaluate a comprehensive method qualification package that includes intra-and inter-run precision and accuracy across all evaluated concentrations, selectivity, recovery and matrix effect, dilution linearity, and carryover. Additionally, we illustrate that this assay provides a rapid and accurate means of monitoring high risk HCP clearance for in-process support and can actively guide process improvement and optimization. Lastly, we compare direct digestion platforms and affinity depletion platforms to demonstrate the impact of HCP−mAb interaction on lipase quantification.
Insertion loss in octave bands was measured for 14 residences in upstate New York using a carefully developed procedure similar to ASTM E336-71 A1.2. Although other dwelling attenuation data are available [e.g., House Noise-Reduction Measurements for Use in Studies of Aircraft Flyover Noise, Soc. Automative Engrs., AIR 1081; Oct. 1971] none was clearly applicable to rural areas of upstate New York and to sources of noise located near the ground. The source was simulated by a three-speaker array using broadband noise. Attenuation of transmission line noise was derived mathematically by applying the measured octave-band attenuations to a typical broadband transmission line noise spectrum and converting to A-weighted and sound levels. Average attenuation was about 15 dBA with windows open 2 ft2, and about 30 dBA with windows closed. [The study was conducted by Rochester Gas and Electric Corporation and their consultant Bolt Beranek and Newman in consultation with the New York State Departments of Public Service and Environmental Conservation.]
The autocorrelation of a series of noise parameters (Leq, L10, L50, or L90) was examined. The noise parameters were derived from 15-min records of ambient noise collected at the same hour (0000, 0600, or 1800 h) on each of 100 (predominantly consecutive) days at a rural site. The same analysis was also performed for a suburban and a quiet, urban, residential site. The first zero crossing (indicating statistical independence) occurs at 9–13 days for the rural site. A run test for randomness (0.05 level of significance) was also used. The random hypothesis consistently failed for the rural site, showing that consecutive days do not produce a random string of data. It is shown that for an efficient ambient survey, samples should be separated by at least three days for rural sites, but that consecutive day samples are normally satisfactory for urban and suburban sites.
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