June 15, 2016 (Vol. 36, No. 12)

Thermo Fisher Scientific’s Protein Characterization Approach Integrates Enzymatic Digestion, Chromatographic Separation, Mass-Spec Detection, and Data Processing

Monoclonal antibodies are the major element in the fastest growing sector of biopharmaceuticals within the pharma industry. In 2016, 8 of the top 10 drugs are therapeutic proteins. Their manufacture is accomplished in bacterial or eukaryotic expression systems, requiring extensive purification of the target product. During drug development and production, the quality of biotherapeutics needs to be closely monitored.

Various analytical methods have been used to study quality attributes such as structural integrity, aggregation, glycosylation pattern, or amino acid modification. Methods based on ultra high-performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HRMS) are among the most powerful protein-characterization techniques.

Proteins can be enzymatically digested to obtain peptides enabling their analysis by means of peptide-mapping experiments. Peptide mapping is valuable since it reveals minor variations that would be difficult or impossible to see at the intact level, and is therefore necessary throughout the discovery, development, clinical, and QA/QC phases of biologic production. In comparison to traditional HPLC, UHPLC delivers much higher peak capacity that allows better resolution of complex mixtures such as protein digests. Moreover, owing to the smaller particle size typically used in UHPLC, the loss in resolving power with flow rate is less pronounced.

The multifaceted, lengthy nature of the peptide-mapping workflow has, to date, been a barrier in the introduction of this information-rich technique for process monitoring.

In this article, fast and sensitive approaches combining enzymatic digestion, efficient chromatographic separation, HRMS, and rapid data processing within the high-throughput, biopharmaceutical environment are discussed.

Materials and Methods

The commercially available monoclonal antibody rituximab (F. Hoffmann-La Roche) was digested using the Thermo Scientific SMART Digest™ kit. The sample was diluted 1:4 with the SMART digestion buffer included in the kit, and enzymatic digestion was allowed to proceed at 70°C for 45 min at 1400 rpm on a shaker. Disulfide bonds were reduced after the digestion by incubation for 30 minutes at room temperature with 5 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP).

A Thermo Scientific Vanquish™ UHPLC system with a 2.1 × 250 mm Thermo Scientific Acclaim™ Vanquish C18, 2.2 μm column and gradients of water and acetonitrile (ACN) with 0.1% formic acid (FA) each were used to separate the peptide mixtures. Five different separation times were applied and compared: 5, 8, 13, 20, and 30 min for the gradient ramping from 4% to 55% eluent B (0.1 % FA in 8:2 ACN/water (v/v)).

Flow rates were adapted accordingly using 1.1 (5 min), 1.0 (8 min), 0.6 (13 min), 0.4 (20 min), and 0.4 mL/min (30 min). The Thermo Scientific Q Exactive™ HF mass spectrometer (MS) equipped with a HESI-II probe was used for mass spectrometric detection using a full MS/dd-MS2 (Top 5) experiment.

The data were acquired with the Thermo Scientific Dionex™ Chromeleon™ Data System, version 7.2 SR4, and Thermo Scientific BioPharma Finder™ software, version 1.0, was used for data analysis.

Rapid Peptide Maps

Peptide-mapping experiments were performed to assess the sequence coverage of the light and heavy chains, as well as to identify and (relatively) quantify post-translational modifications (PTMs) such as oxidation, glycosylation, and deamidation. For all five gradient times, from 30 min down to 5 min, excellent separation was achieved and resulting sequence coverages of 100% were obtained from all separation times both for light and heavy chain, even for the very short gradient of 5 min (Figure 1).

A fundamental requirement for peptide-mapping workflows is reproducibility. This enables users to confidently assign data differences to the sample and not the methodological conditions employed. Reproducibility is influenced by protein digestion, chromatographic separation, detector performance, and linearity and consistency in data handling.

Recent advances in UHPLC instrumentation and chemistries have significantly improved the robustness and efficiency of proteins and peptide separations. When combined with simplified kit-based protein digest procedures incorporating optimized, heat-stable trypsin, the issues of irreproducibility that are often associated with in-solution protein digestion are eliminated.

The resultant digest is highly reproducible, less prone to chemically induced PTMs, autolysis-free and highly amenable to automation.

Data handling/processing is also an important consideration for the incorporation of such a workflow into a high-throughput environment. The software package used to control the instrumentation and acquire the data should be GxP compliance-ready and a sophisticated yet simple software package can expedite data interpretation.

The aim of a peptide-mapping experiment is twofold: firstly, to obtain 100% protein sequence coverage via detection of the constituent peptides; secondly, to confirm the protein primary sequence and site-specific quantitative information on the PTMs within the protein.

In a HRMS peptide-mapping experiment MS1 data reveals the precursor ion masses of the peptides and MS2 data reveals the b- and y-ion amino acid fragments of each peptide, and thus information on the modifications that are present. Even with ultrashort gradients, as shown in Figure 1, spectacular separation efficiency and 100% coverage can still be obtained.

Typically, 6 Full MS1 spectra and 25 (5 × Top 5) MS2 spectra were acquired for these peaks. This type of MS scan speed is key to the success in obtaining full sequence coverage, and therefore in fully understanding the end product.

Figure 1. Total ion chromatograms obtained from peptide-mapping experiments of rituximab applying gradient lengths 30, 20, 13, 8, and 5 min. Flow rates and resulting pressures are indicated in the individual traces.


The workflow and data presented clearly demonstrate the capability of the applied UHPLC-HRMS setup to significantly speed up peptide-mapping experiments, thus enabling high-throughput analyses, for example, during clone selection in the development phase of biopharmaceuticals, during QA/QC and for at-line process monitoring.

Obtaining detail at the amino acid level within short (minutes to hours) timescales during bioprocessing will enable important adjustments to be made in production and ensure the highest quality product is produced. This is of particular importance in the production of biosimilars, where product quality often takes precedence over quantity and preservation of profitability is paramount.

Rowan Moore ([email protected]) is vertical marketing manager for pharmaceutical and biopharmaceutical HPLC and IC and Martin Samonig is a biopharmaceutical HPLC and MS applications scientist at Thermo Fisher Scientific.

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