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Routine Analysis of Host Cell Proteins in Antibody Preparations using PASEF
Poster Title: Routine Analysis of Host Cell Proteins in Antibody Preparations using PASEF
Submitted on 13 Feb 2019
Author(s): Stuart Pengelley1, Guillaume Tremintin², Waltraud Evers1, Detlev Suckau1
Affiliations: 1Bruker Daltonik GmbH, 28359 Bremen, Germany 2Bruker Daltonics, Billerica, USA
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Poster Information
Abstract: Methods
The NISTmAb Reference Material 8671 and the Universal Proteomics Standard (UPS1, Sigma) were reduced using DTT in TFE solution and alkylated with iodacetamide prior to overnight digestion with trypsin (Promega). Peptides were separated on an Intensity Solo 2 1.8μm C18 100 x 2.1 mm column using an Elute UHPLC coupled to a timsTOF Pro ion mobility QTOF mass spectrometer (all Bruker Daltonics). A 150 minute gradient was used in a total runtime of 165 minutes. For nanospray, a nanoElute UHPLC was fitted with an IonOpticks 25 cm x 75 μm 1.6 μm C18 column using a 210 minute gradient. PASEF scans were recorded and searched against the mouse SwissProt database using Mascot.

Results: Standard flow UHPLC
The UPS1 standard was used to make a 5-step 1:3 dilution series in a constant background of NIST mAb over a concentration range from 0.3 to 934 ppm. Each dilution was measured in triplicate and the amount of NIST mAb loaded on column was 25 μg for each injection. PASEF enabled the detection of UPS1 proteins down to low single digit ppm concentrations in the presence of 25 μg NIST mAb. Figure 2 shows the linear response for the UPS1 protein beta-2-microglobulin in the concentration range from 132 ppm to 1.6 ppm.
The dilution series indicated that PASEF enabled detection of HCPs in the range of 1 to 100 ppm of the therapeutic protein, which usually requires special approaches such as 2D-LC or the use of libraries. In a model experiment, 30 μg NIST mAb was loaded onto the column to measure HCPs in the NIST mAb sample. This resulted in the detection of 8 proteins with 3 or more peptides and a further 17 proteins with 2 peptides, at 1 % FDR (Fig. 3). These results illustrate the use of PASEF to identify HCPs using a routine 1D-UPLC-MS configuration.

Results: nano flow UHPLC
A standard proteomics setup using nano UHPLC was also evaluated for its suitability for HCP identification. This setup has already been established as the new benchmark for
bottom-up proteomics applications (1, 2). In comparison to other nano LC setups, robustness is improved by the CaptiveSpray ion source, which allows ions to be sprayed directly from the emitter for increased sensitivity. Figure 4 shows that > 200 HCPs were identified in 1.5 μg NIST mAb, including expected and previously unreported HCP proteins (Fig. 5). As shown in Figure 6 the depth of HCP coverage and sequence coverage was further extended to >280 HCP identifications by employing an alternate digestion method in which only the HCPs are digested allowing the mAb to be removed prior to analysis (3).
Summary: In this work we show how PASEF (parallel accumulation and serial fragmentation) scans, as implemented on the timsTOF PRO QTOF (Fig 1), can be applied to HCP analysis to achieve the goal of sensitive detection with enhanced speed and data quality.References: (1)Meier et al.; J Proteome Res. 2015 Dec 4;14(12):5378-87
(2)Lubeck et al.; Bruker Application Note 131
(3)Huang et al.; Anal. Chem. 2017, 89, 5436-5444
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