FlashNoteHigh Throughput HCP Screening is here:> 50 HCPs identified in 21 minutes
Monitoring of host cell proteins (HCPs) for biotherapeutics is required during
manufacture and prior to product release. Bottom-up mass spectrometry
approaches are established for analysing HCPs, but the low abundance of manyof these proteins presents a significant challenge for confident detection ofHCPs, especially when using short gradients. Here, the high speed of PASEF,(parallel accumulation and serial fragmentation) as implemented on thetimsTOF Pro, coupled with the Evosep One LC system were used to detect
HCPs using a 21 minute gradient.

Challenge
HCPs are present in biotherapeutic drug preparations, often at low ppm concentration levels, and must be
monitored due to stability, efficacy and immunogenicity concerns. Sensitive detection of HCPs usually requires
high sample amounts to be loaded on the HPLC column which are then separated using a long elution gradient.
The resulting sample-to-sample times are not suitable for routine high-throughput monitoring of HCP removal
during development and manufacture of biotherapeutics.

Solution
PASEF is a novel data-dependent method for MS/MS fragmentation
at spectra acquisition rates up to 120 Hz which features automaticre-sampling of low abundant precursors, generating high quality
MS/MS spectra with high sensitivity. The speed of this instrument
is a perfect fit with the Evosep One, which efficiently delivers shortgradients using single use and disposable Evotip trap-columns toprevent carryover between samples and ensures reliable data fromeach analysis. This system provides high sample turnarounds of
60 samples per day with a 21 minute gradient for HCP analysis.
Fast, High-Throughput, High Confidence HCP Analysis!
timsTOF Pro and Evosep One combine to provide a fast, sensitiveand straight-forward workflow for high-throughput HCP analysis.

Authors: Stuart Pengelley, Heiner Koch, Thomas Kosinski, Eckhard Belau; Bruker Daltonik, Bremen

MethodDigestion
Twenty five µg NISTmAb (NIST Reference Material 8671, Humanized IgG1kMonoclonal Antibody) was digested
with Trypsin (Promega) according tothe protocol published by Huang et al.
(Anal. Chem. 2017, 89, 5436−5444). In
this method reduction is not performed
prior to digestion, the mAb remains
largely intact and is removed prior toLC-MS/MS by precipitation.
Figure 1: HCPs identified in 21 minutes with 3 or more peptides

LC-MS/MS

Data were acquired on the timsTOF Pro
applying a 0.5 sec cycle consisting of 1
TIMS MS scan and 4 PASEF MS/MSscans. PASEF scans were searchedagainst the mouse SwissProt database
at 1% FDR using PEAKS Studio 8.5(Bioinformatics Solutions Inc.).

Results
61 HCPs were identified using a 21
minute gradient, 28 with two or morepeptides. Example MS/MS spectra areshown which matched to adenylatekinase 2, mitochondrial, a NISTmAb
HCP reported at a concentration of2 ppm (Huang et al., 2017).

Figure 2: MS/MS Spectra for adenylate kinase 2, mitochondrial

For research use only. Not for use in diagnostic procedures.

Bruker Daltonics GmbH & Co. KG

Bruker Scientific LLC

Bremen · Germany
Phone +49 (0)421-2205-0

Billerica, MA · USA
Phone +1 (978) 663-3660

ms.sales.bdal@bruker.com – www.bruker.com

You are looking for further Information?
Check out the Link or scan the QR Code.
www.bruker.com/timstofpro

Bruker Daltonics is continually improving its products and reserves the rightto change specifications without notice. © Bruker Daltonics 09-2018, FN-006, Rev. 01

The sample was loaded onto an Evotip.
This serves as a disposable pre-column
for the Evosep One, which features
Gradient Offset Focussing™. Using
this method the separation gradient is
21 minutes with a total runtime of 24
minutes, allowing 60 samples to bemeasured per day.