Thanks to their high target specificity and long half-life in humans, therapeutic monoclonal antibodies (mAbs) have grown steadily in use to become the leading biotherapeutic product class.1 Over the past three decades, more than 80 mAbs have received regulatory approval for the treatment of a broad range of diseases, including inflammatory, autoimmune and cardiovascular conditions as well as cancer.2,3 The therapeutic impact of mAbs is set to further increase, with the market expected to grow by almost 7% annually to reach an estimated US$174 billion by 2026.4
Like many other biotherapeutic products, mAbs are large, highly complex molecules that are manufactured using advanced synthesis and purification processes. Given this complexity, rigorous characterization techniques are essential to monitor mAb critical quality attributes (CQAs) and ensure the release of safe and effective product batches. These methods must be capable of screening for a broad range of impurities and post-translational modifications (PTMs) potentially introduced during production.
Peptide mapping remains the ‘gold standard’ approach for mAb characterization. The technique, which involves digesting mAbs using enzymes or chemical reagents to produce peptide fragments that are subsequently separated using liquid chromatography and analyzed by mass spectrometry, is widely used for primary structure identification, as well as quality control (QC) and quality assurance (QA) measures.
Various peptide mapping approaches have been developed. However, protein digestion protocols have consistently suffered from a lack of reproducibility, resulting in poor confidence in results. Commonly- used trypsin-based digestion protocols also typically involve costly, time-consuming and inefficient sample handling procedures, as well as long digestion times of several hours. Additionally, many of the sample preparation methods employed in traditional peptide mapping workflows can result in a wide range of sample preparation-induced PTMs, and these steps are often not amenable to automation, limiting their throughput and speed.
To support large-scale production processes and accelerate the delivery of safe, high-quality mAb products, it is essential that peptide mapping workflows are simple, robust and reproducible.
Advancing Peptide Mapping Workflows using Digest Kits
Recent advances in trypsin-based protein digestion protocols are simplifying peptide mapping workflows and improving method reproducibility. In turn, these advances are helping to increase throughput and enhance confidence in results. The development of commercial digest kits that include thermally stable, immobilized trypsin has enabled high-temperature protein denaturation without the need to add denaturants, resulting in faster, easier and more consistent protein digestion. Some of these kits also support automated sample preparation and digestion workflows, further accelerating peptide mapping approaches by eliminating error-prone manual steps, increasing throughput and enhancing confidence in results.
The ease of use, robustness and reproducibility of modern trypsin digest kits were recently investigated in a comparative study. Four tryptic digestion protocols were evaluated: a traditional overnight in-solution digestion method using heat for protein denaturation, an alternative rapid digestion protocol,5 and two commercially available digest kits, the Thermo Scientifi c SMART Digest Trypsin Kit and the Thermo Scientific SMART Digest Trypsin Kit with Magnetic Bulk Resin option. As part of this last protocol, the magnetic bead-based digest kit was used in combination with an automated purifi cation system.
Two mAbs were used to investigate these four digestion protocols: the commercial biotherapeutic product adalimumab, and a reference standard, NISTmAb Reference Material 8671 (herein referred to as NISTmAb), which is commonly used to evaluate the performance of analytical methods. The samples were analyzed by ultra-high throughput liquid chromatography (UHPLC) in combination with mass spectrometry.
Sample Preparation and Pretreatment
Adalimumab was obtained from a commercial supplier at a concentration of 50 mg/mL. NISTmAb was provided by the National Institute of Standards and Technology at a concentration of 10 mg/mL. For each of the four digestion protocols, mAb samples were prepared in triplicate by two different analysts over three days.
Digestion Protocols
In-solution digestion protocol using heat denaturation: Samples were diluted to 2 mg/mL in water. The reduction of disulfide bonds was carried out by the addition of 10 mM dithiothreitol (DTT) in 100 mM triethylammonium bicarbonate (pH 8.5), followed by incubation at 70°C for 75 minutes. Alkylation was carried out by the addition of 20 mM iodoacetic acid (IA). The samples were kept in darkness for 30 minutes, before 11 mM DTT was added to quench the IA. Trypsin was added to the mAb samples at a protein–trypsin ratio of 1:50, and tryptic digestion was performed at 37 °C for 16 hours, before being quenched with 10% trifl uoroacetic acid (TFA).
Alternative rapid digestion protocol: A peptide mapping method described by Rogers et al.5 was followed. Samples were diluted to 2 mg/mL, denatured, and reduced using 10 mM DTT in 7.5 M guanidine (pH 8.3) at ambient temperature for 30 minutes. The samples were alkylated by the addition of 20 mM IA and incubated in darkness for 20 minutes, before being quenched with 11 mM DTT. Sample solutions were desalted using BioRad BioSpin P6 columns and eluted in 100 mM tris buffer (pH 8.0). Protein concentration was measured with a Thermo Scientific NanoDrop 2000 spectrophotometer. Trypsin was added to the mAb samples at a protein–trypsin ratio of 1:10, and tryptic digestion was performed at 37 °C for 30 minutes, before being quenched with 10% TFA.
SMART Digest Trypsin Kit protocol: Samples were diluted to 2 mg/mL in water. 150 μL of SMART Digest trypsin buffer was added to the SMART Digest vials, followed by 50 μL of sample (100 μg). mAb samples were incubated at 1400 rpm at 70 °C for 45 minutes. Following tryptic digestion, samples were spun down for 2 minutes at 7000 rpm and the supernatant was transferred to a second tube. The reduction of disulfide bonds was carried out by the addition of 10 mM DTT, followed by incubation at 57 °C for 30 minutes.
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SMART Digest Trypsin Kit with Magnetic Bulk Resin option protocol: Samples were diluted to 2 mg/mL in water. For each sample digest, sample and buffers were added to each lane of a Thermo Scientific KingFisher Deepwell 96-well plate. Bead wash buffer was prepared by diluting the SMART Digest buffer 1:4 (v/v) in water. Neat SMART Digest buffer was used as the bead buffer. Digestion was carried out using a Thermo Scientific KingFisher Duo Prime purification system according to the protocol described in Table 1. Samples were incubated at 70 °C for 45 minutes on medium mixing speed to prevent sedimentation of the beads, and subsequently cooled to 10 °C postdigestion. Disulfide bond reduction was carried out with 10 mM DTT at 57 °C for 30 minutes, and alkylation was performed with 20 mM IA in darkness for 30 minutes. The reaction was quenched with 11 mM DTT followed by the addition of 10% TFA.
Analytical System and UHPLC Conditions
The analytical setup consisted of a Thermo Scientific Vanquish Flex Binary UHPLC system, in combination with a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer.
Samples were separated by UHPLC using a gradient method with a total run time of 65 minutes. Mobile phase A consisted of water with 0.1% formic acid (v/v). Mobile phase B consisted of acetonitrile with 0.1% formic acid (v/v). A flow rate of 0.3 mL/min was used, with an injection volume of 10 μL. The column was maintained at a temperature of 25 °C.
Simple, Rapid and Reproducible Protein Digestion
Analysis of the digested mAbs highlighted the excellent reproducibility of the UHPLC method. Chromatograms for six sample preparations of NISTmAb, using the four protein digestion methods, are shown in Figure 1.
For nearly all protein digestion experiments, 100% coverage was achieved, as shown in Table 1. Greater than 99% coverage was achieved for the adalimumab heavy chain peptides. Notably, the rapid digest protocol and the two digest kit protocols were capable of complete sequence coverage using digestion times of less than 45 minutes.
Certain aspects of sample preparation and digestion may induce PTMs such as the deamidation and oxidation of amino acid residues. To evaluate the impact of each of the sample digestion protocols on the number of PTMs, the percentage relative abundance of modified peptides was determined for both mAbs. The average percentage relative abundance of deamidation and oxidation modifications for adalimumab (Figure 2) and NISTmAb (Figure 3) are shown.
The in-solution, heat digestion protocol resulted in the highest levels of deamidation and oxidation for both adalimumab and NISTmAb. The digest kits and alternative rapid digest protocol generally resulted in lower levels of deamidation and oxidation than the in-solution, heat digestion method. Interestingly, adalimumab resulted in higher total levels of modifications than NISTmAb for all studied digestion methods.
Studies have shown that high buffer pH and long digestion times can result in higher levels of deamidated peptides.6 In general, the results presented here are consistent with these findings, as the in-solution, heat digestion protocol, with its relatively high pH 8.5 and 16-hour digestion, resulted in more deamidated peptides than the two digest kit methods, which were performed at a milder pH and required just 45 minutes for digestion. Some deamidated peptide forms, such as N318 and N319 for NISTmAb and adalimumab respectively, were around two-fold higher using the in-solution, heat method than when the digest kits were used.
Higher levels of methionine oxidation were also detected following sample processing using the in-solution, heat digestion protocol than using the alternative rapid digestion method and digestion kits. The M256 modified peptide, for example, was approximately three-fold higher when the in-solution, heat digestion protocol was employed compared to the other methods.
The sample handling requirements and number of preparation steps involved in each of the digestion protocols varied significantly. The use of guanidine in the alternative rapid digest protocol, for example, necessitated the use of a desalting step in the sample preparation process, resulting in a 30% reduction in sample recovery. The automated magnetic bead-based digest kit method, on the other hand, required minimal manual intervention. Protocols based on multiple manual preparation steps can lead to inconsistencies in sample handling and digestion conditions, resulting in greater variation in the number of sample preparation-induced PTMs and reduced confidence in results.
To assess the reproducibility of the four sample preparation and digestion protocols, the standard deviation associated with the deamidation and oxidation modifications was investigated. The lowest standard deviation values for modified peptides were recorded for the samples digested using the digest kits, highlighting the improved consistency afforded by these methods. The excellent reproducibility, ease of automation, and low levels of sample preparation-induced PTMs associated with the SMART Digest kits highlights the suitability of these two methods for high-throughput environments where the confident characterization of biotherapeutics is essential.
Conclusion
Peptide mapping is widely regarded as the method of choice for mAb characterization. However, the poor reproducibility of many traditional digestion protocols and potential for mAb deamidation and oxidation modifications are major challenges. Modern digestion kits are simplifying sample digestion protocols, shortening digestion times, and minimizing sample preparation-induced PTMs to deliver enhanced levels of method reliability and consistency. By driving confidence in results, these more effective peptide mapping methods are set to accelerate the delivery of innovative mAbs and other biotherapeutics to patients.
Author Biographies
Tom Buchanan joined Thermo Fisher Scientific in 2018 as a European Bio/Pharma Applications Development Scientist from Allergan Biologics where he was a Biopharmaceutical Characterization Scientist within Analytical Development. At Allergan he was responsible for the development of LC-UV/LC-MS peptide mapping methods for biologics, in particular, monoclonal antibodies, to determine critical quality attributes and intact mass methods for product identification. Tom has expertise in cGMP downstream bioproduction and quality control, as well as in-process batch testing, stability studies, and analytical method validation.
Dr. Sara Carillo is the applications development team leader in the Characterization and Comparability Laboratory in NIBRT, Ireland. Sara completed her PhD in Chemical Science at the University of Naples in 2013 working on bacterial glyco-conjugates structural characterization. In 2015, Sara joined Dr. Jonathan Bones’s research group in NIBRT, working on CHO cell glycome and biotherapeutics characterization.
Jonathan Bones received his PhD in Analytical Chemistry from Dublin City University in 2007. Jonathan then moved to NIBRT, working under the mentorship of Prof. Rudd. In 2010, Jonathan was appointed the John Hatsopoulos Research Scholar within the Barnett Institute, Boston, working under the mentorship of Prof. Karger. Jonathan returned to NIBRT in 2012 and is the PI of the Characterization and Comparability Laboratory and an Associate Professor in the School of Chemical and Bioprocess Engineering at University College Dublin.
References
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- Animal Cell Technology Industrial Platform. Monoclonal Antibodies Approved by the EMA and FDA for Therapeutic Use (status 2017). http://www.actip.org/products/monoclonalantibodies-approved-by-the-ema-and-fda-for-therapeutic-use. Updated May 2017. Accessed June 2019.
- Coherent Market Insights. Monoclonal Antibody Therapeutics Market – Size, Share, Outlook, and Opportunity Analysis, 2018-2026. https://www.coherentmarketinsights.com/market-insight/monoclonal-antibody-therapeutics-market-2403. Published January 2019. Accessed June 2019.
- Rogers RS, Nightlinger NS, Livingston B, Campbell P, Bailey R, Balland A. Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics. mAbs. 2015; 7(5): 881–890.
- Ren D, Pipes GD, Liu D, et al. An improved trypsin digestion method minimizes digestioninduced modifications on proteins. Anal. Biochem. 2009; 392(1): 12–21.