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www.biopharminternational.com July 2021 BioPharm International eBook 27 which gradually break down the ther- apeutic protein or excipient, affecting the stability of the product over time. HCPs are also capable of mimicking the action of therapeutic proteins in assays, possibly resulting in the mis- formulation of the product outside the therapeutic window (3). Removing HCPs from biologic prod- ucts is vital for protecting drug efficacy, stability, and safety. During biothera- peutic production, the drug product is separated from impurities using chro- matography and the remaining product analyzed for residual HCPs. However, even after several purif ication steps, low-level residual HCPs may remain. Although there is no specific guidance from regulatory agencies relating to the exact acceptable limit of HCP in a prod- uct, biotherapeutics are typically expected to contain less than 100 ng HCP/mg of product. T he 'gold sta nda rd ' techn iques for HCP analysis are immunoassays such as enzyme-linked immunosor- bent assays (ELISA). Although highly sensitive, high-throughput methods, immunoassays have long lead times and can be expensive to set up. In addition, ELISAs only measure total HCP and do not support the identifi- cation of individual HCPs. This, and the complexity and diversity of HCPs, necessitates a more powerful tool for analyzing these impurities. INCREASING COVERAGE OF LOW ABUNDANT PEPTIDES Mass spectrometry (MS) is now recog- nized as an ideal orthogonal method to immunoassays for HCP analysis. The faster set-up time of MS meets the evolv- ing needs of biopharma labs with chang- ing bioprocesses and allows scientists to rapidly monitor and identify multi- ple protein analytes in the same sam- ple. Furthermore, the ability of MS to detect very low amounts of HCP in a non-targeted manner is crucial, given that even trace levels of HCP can cause immunogenicity and impact drug qual- ity. Advanced MS techniques not only monitor, but characterize several impu- rities using a single method, bringing the high discriminatory power needed to separate impurities while providing high sensitivity analysis to detect and quantify low-level HCPs. A cha l lenge facing MS ana lysis arises from the vast abundance of prod- uct-derived peptides relative to impu- rity peptides. One approach to address this issue of high dynamic range and potential ion suppression of the HCP peptides is to apply liquid chroma- tography tandem mass spectrometry (LC–MS–MS) analysis to reduce the simultaneous product contribution to total ions in the mass spectrometer, the effect of which can be further improved if partially resolved HCP preparations are used (4). In this manner, LC–MS– MS can be employed as an import- ant tool for monitoring specific critical HCPs and supporting risk assessment for biopharmaceutical products. Another approach to analyze peptides over the wide dynamic range required for HCP characterization is by using ion mobility. Sophisticated modern mass spectrometers implementing ion mobility separation can identify low-level HCPs with high confidence and achieve deep coverage, without compromising speed. For example, trapped ion mobility spec- trometry (TIMS) coupled with quad- rupole time-of-flight (QTOF) MS can significantly increase 'chromatographic' resolving power for peptide analysis— vital for the analysis of samples contain- ing HCPs. The TIMS focusing and subsequent mobility separation occurs in the gas phase, adding the second stage of separation to LC–MS, while increasing sensitivity and confidence in compound characterization. Combined with the fast acquisi- tion speeds (>100 Hz MS–MS) of the parallel accumulation serial frag- mentation (PASEF) method, TIMS QTOF-MS–MS technology enables high sequencing speeds while generat- ing high-quality spectra from complex samples (5). Almost 100% duty cycle can be achieved, meaning almost every ion that enters the mass spectrometer is mea- sured—thus maximizing the use of all ions, which is ideal for identifying low abundant peptides. When coupled with an advanced high-performance liquid chromatography (HPLC) system such as the Evosep One (Evosep, Denmark), TIMS QTOF-MS–MS with PASEF can routinely detect HCPs using short gradients, for example the 21-minute gradient, which permits 60 samples per day to be analyzed (6). The resulting sensitivity and speed increase is important in both upstream and downstream processing, respectively. Upstream analysis In early process development, critical problems can arise in production or sta- bility testing. Upstream HCP analysis requires longer runs, typically 90 minutes or more, for increased depth of analysis and maximum sensitivity. Identifying potentially troublesome HCPs as early as possible in the development process is vital. If a HCP that is difficult or impossible to remove is identified further downstream, there is a greater financial risk given the considerable upfront devel- opment costs. Downstream analysis Rapid analysis of downstream pro- cesses, including quality control (QC) and batch release, requires fast runs and Biopharmaceutical Analysis Impurity Analysis A challenge facing mass spectrometry analysis arises from the vast abundance of product-derived peptides relative to impurity peptides.