一种高通量单克隆抗体肽图Mapping的LC-MS方法
High-Throughput Peptide Mapping of Monoclonal Antibodies Using Tandem Liquid Chromatography– Mass Spectrometry
Analytical strategies for high quality management (QC) of monoclonal antibodies (mAbs) depend on peptide mapping workflows with liquid chromatography–mass spectrometry (LC–MS) methods. While LC–MS is efficient, peptide mapping with LC–MS can contain lengthy evaluation occasions that cut back the throughput of QC testing. Fashionable tandem LC strategies can improve evaluation effectivity and are designed to scale back the downtime of MS sampling to extend the effectivity of QC pipelines. The method offered right here improves the effectivity of mAb evaluation peptide mapping, while returning high-quality information.
【机翻】单克隆抗体(mAbs)的高质量管理(QC)的分析策略依赖于液相色谱-质谱(LC-MS)方法的肽图谱工作流程。虽然LC-MS是高效的，但使用LC-MS进行肽图谱绘制可能会包含冗长的评估场合，从而降低QC测试的吞吐量。时尚的串联LC策略可以提高评估效率，并被设计为缩减MS采样的停机时间，以延长QC管道的有效性。这里提供的方法提高了单抗评价肽图的有效性，同时返回了高质量的信息。
The event and manufacturing of therapeutics, comparable to monoclonal antibodies (mAbs), require high-throughput pattern evaluation and excessive‑high quality information. For instance, post-translational modifications (PTMs) can have a serious affect on the standard and efficacy of the ultimate product and, due to this fact, should be monitored. Orthogonal analyses of protein PTMs are required to make sure sure crucial high quality attributes (CQAs) are inside the established ranges to take care of lot-to-lot consistency in keeping with regulatory necessities and Worldwide Council for Harmonisation of Technical Necessities for Prescribed drugs for Human Use (ICH) tips (1).
Peptide mapping is taken into account a gold normal for mAb evaluation as a result of it delivers data on many attributes on the peptide stage inside a single evaluation, offering a powerful methodology for an analytical management technique. One kind of peptide mapping method utilizing liquid chromatography–mass spectrometry (LC–MS) evaluation is the multi-attribute methodology (MAM), which permits high-quality, assured PTM identification in analysis and at later phases in high quality management (QC) laboratories (2–4). Nevertheless, peptide mapping with LC–MS strategies can contain lengthy evaluation occasions because of the prolonged LC gradient wanted to resolve peptides within the advanced pattern ensuing from tryptic digestion. This creates challenges for high-throughput laboratories the place effectivity is vital. Furthermore, lengthy downtimes are additionally created between analyses to permit for column washing and re-equilibration steps, and when eluents are diverted to waste. Throughout these durations, no separation is happening. This reduces the time accessible to carry out MS testing and reduces effectivity in analysis and QC pipelines.
Improvements in ultrahigh-performance liquid chromatography (UHPLC) instrumentation have led to the event of a tandem LC system that may be leveraged for high-throughput “tandem-mode” LC–MS evaluation.
This text demonstrates the applicability of a tandem LC–MS workflow for peptide mapping evaluation of trastuzumab in a high-throughput method. With a tandem LC–MS method, a better variety of pattern injections had been carried out in the identical timeframe, rising analytical throughput and considerably decreasing MS downtime. Chromatographic outcomes had been discovered to be equal throughout the 2 chromatographic channels. Moreover, the reproducibility of the obtained PTM values throughout a steady 16-h interval of operation had been examined, demonstrating the soundness of MS efficiency beneath steady acquisition.
Supplies and Strategies
Pattern Preparation: Samples had been ready following a printed methodology (5) to acquire peptides from tryptic digestion of trastuzumab. Trastuzumab samples had been diluted to 2 mg/mL in water. For every pattern digest, pattern, digestion buffer (buffer 1, pH 6.5 or buffer 2, pH 7.2), and 5 mM TCEP (closing focus) had been added to every lane of a Thermo Scientific KingFisher deep properly 96-well plate. Trypsin bead “wash buffer” was ready by diluting digestion buffer 1:4 (v/v) in water. Bead buffer was neat digestion buffer. Digestion was carried out utilizing a Thermo Scientific KingFisher Duo Prime Purification System with Thermo Scientific BindIt software program (model 4.0). Samples had been incubated for five to 40 min at 70 °C on medium mixing pace to forestall sedimentation of beads for the digestion time course research, and beads had been eliminated at every time level. Following digestion, 100-μL samples had been transferred to 300‑μL Thermo Scientific PP Screw vials and cap and 1 μL of 10% trifluoroacetic acid (TFA) was added (closing focus 0.1% TFA) and instantly analyzed by high-resolution correct mass LC–MS.
Chromatography Step: The resulted peptides had been separated on a 2.1 × 250 mm, 2.2‑μm Acclaim Vanquish C18 column (Thermo Scientific) utilizing the Thermo Scientific Vanquish Horizon Duo UHPLC System for tandem LC–MS workflows. Evaluation was carried out utilizing a binary gradient of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B) with a move fee of 0.3 mL/min. The gradient began at 2% B and elevated to 40% B over 45 min on the analytical pump, whereas the reconditioning pump carried out two cycles from 2% to 80% B in 20 min and equilibrated again to 2% B for 25 min.
Mass spectrometry information acquisition was carried out with the Thermo Scientific Orbitrap Exploris 240 MS system with biopharma choice at decision 120,000 FWHM. Utility‑particular MS tune and acquisition settings are templated and supplied inside the Thermo Scientific Chromeleon Chromatography Information System (CDS) software program, which makes this method immediately and simply transferable from instrument to instrument.
Tune Settings: Spray voltage: 3.8 kV; sheath gasoline: 25; auxiliary gasoline: 10; ion switch tube temperature: 320 °C; vaporizer temperature: 150 °C; strain mode: peptide.
MS Scan Settings: Decision: 120,000 (at m/z200); scan vary: 200–2000 m/z; RF lens (%): 60; AGC goal: 3 × 106; max injection time: 100 ms.
MS/MS Settings: Decision: 15,000; normalized collision vitality: 30; injection time: 200 ms; AGC goal: 1 × 106; TopN: 5; microscan: 1; isolation window: 2 m/z.
Information had been processed utilizing the Thermo Scientific BioPharma Finder Software program 4.0.
Outcomes and Dialogue
Peptide mapping is a routine evaluation in biopharmaceutical evaluation and is gaining extra curiosity as a part of the MAM workflow. It’s driving the broader adoption of MS strategies for later phases of biopharmaceutical product growth and, finally, permitting its use within the QC laboratory. Methodology switch onto the QC stage requires improved robustness and excessive‑throughput because of the excessive variety of samples that have to be processed and the required reliability of the strategies assuring product high quality.
On this research, trastuzumab monoclonal antibody was digested utilizing a trypsin protein digestion package and the technology of tryptic peptides was carried out routinely on the purification system. The digested monoclonal antibody was then analyzed by way of tandem
LC–MS/MS evaluation. CDS software program routinely cut up the gradient throughout the 2 columns and pumps, managing when the analytical gradient was full and the wash step began. Twenty‑one pattern injections had been carried out on this research, alternating between the 2 columns to acquire an ideal overlap of runs coming from the 2 channels (Determine 1). This qualitative analysis demonstrates the reproducibility of LC efficiency not solely throughout the 2 channels but additionally when utilizing two completely different columns.
Chromatographic reproducibility was evaluated throughout the gradient by plotting the retention occasions for 5 completely different peptides together with the 21 injections. No distinction was noticed between the 2 information units, returning general relative normal deviation (RSD) values decrease than 1.5% (Determine 2). Wonderful retention time reproducibility is necessary for proper identification of elements to guage and quantify drug product CQAs utilizing MS. Nevertheless, it will be potential to permit larger values of %RSD for the retention occasions when utilizing high-resolution MS, as this stage of decision offers a assured identification of elements even when utilizing a wider retention time window.
Peptide mapping evaluation is principally carried out to guage PTMs in therapeutic proteins that should be monitored throughout bioprocessing and batch launch to ensure drug product high quality. Because of this, we evaluated product high quality attributes for trastuzumab throughout the 21 injections carried out in tandem mode (Desk 1). Every modification was calculated contemplating as much as one missed cleavage, and with out contemplating Na+ and Okay+ adducts or non-specific cleavages, utilizing a confidence rating ≥ 95% and inside ± 5 ppm. Detection of most PTMs resulted in very low RSD values, which exceeded 15% just for some low ample modifications (N328 + deamidation = 0.20%). This proved wonderful chromatographic equivalence of the 2 channels for PTM analysis, in addition to the suitability of the tandem LC–MS workflow for PTMs evaluation and quantitation.
Conclusion
On this research, we’ve demonstrated the suitability of a tandem LC system for tandem LC–MS evaluation for peptide mapping and the MAM workflow. This tandem LC method considerably elevated analytical throughput by decreasing the time when MS was not absolutely utilized for useful information technology; this was achieved by minimizing the time window the place LC was diverted to waste and the MS not utilized. This workflow proved to be a extra time-efficient different to conventional strategies, whereas additionally sustaining or enhancing the efficiency and reproducibility required for a technique to be thought-about for adoption in a QC laboratory.
Twenty-one samples of trastuzumab tryptic digest had been analyzed utilizing LC–MS/MS evaluation in tandem mode. System efficiency was evaluated by monitoring retention time reproducibility and figuring out PTMs, with each evaluations yielding wonderful information and low %RSD values. Tandem evaluation of peptide mapping replicates didn’t have an effect on the accuracy of the outcomes and the right identification, however it had the good thing about saving no less than 6 h of instrument time when in comparison with single mode throughout 24 h. Using automated digest kits versus in-solution digestion has been proven to avoid wasting between 2–20 h preparation time for every pattern (6), offering important pattern preparation time financial savings, which, when mixed with the effectivity advantages of the tandem evaluation, can dramatically improve throughput.
In conclusion, tandem LC–MS represents a strong approach to improve laboratory productiveness with out compromising information high quality, particularly when mixed with automated pattern preparation of the tryptic digest. The operational simplicity of tandem LC–MS strategies implies that little coaching is required for high-throughput peptide mapping of biopharmaceuticals, enabling easy compliance-ready enterprise acquisition of LC–MS information for present good manufacturing follow (cGMP) environments.
【机翻】在这项研究中，我们已经证明了串联LC系统在串联LC - ms评估肽图和MAM工作流中的适用性。这种串联LC方法通过减少MS不能完全用于有用的信息技术时的时间大大提高了分析吞吐量;这是通过最小化LC被浪费和MS未被利用的时间窗口来实现的。这个工作流程被证明是一个与传统策略不同的额外的时间效率，而额外的维持或提高效率和再现性需要一个技术被考虑在QC实验室采用。……（略） 总之，串联LC-MS是一种提高实验室生产效率的有效方法，在不影响高质量信息的情况下，特别是在与胰蛋白酶的自动模式制备混合时。串联LC-MS策略的操作简单性意味着对生物制药的高通量肽图谱需要很少的指导，使符合条件的企业能够容易地获取当前良好的cGMP环境的LC-MS信息。
References
FDA-CDER-CBER, Steering for Trade: High quality Issues in Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product (April 2015).
S. Rogstad, A. Faustino, A. Ruth, D. Keire, M. Boyne, and J. Park, J. Am. Soc. Mass Spectrom. 28, 786–94 (2017).
R.S. Rogers, N.S. Nightlinger, B. Livingston, P. Campbell, R. Bailey, and A. Balland, Mabs 7, 881–90 (2015).
R.S. Rogers, M. Abernathy, D.D. Richardson, J.C. Rouse, J.B. Sperry, P. Swann, et al., The APPS Journal 20, 1–8 (2018).
S. Millán-Martín, C. Jakes, S. Carillo, et al., Anal. Bioanal. Chem. 412, 6833–6848 (2020).
Thermo Scientific Utility Observe 72141, SMART Digest in comparison with basic in-solution digestion of rituximab for in-depth peptide mapping characterization, http://instruments.thermofisher.com/content material/sfs/brochures/AN-1159-SP-SMART-Digest-Peptides-AN72141-EN.pdf
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