Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler Jason Richardson, Bhavana Shah, Gang Xiao, Pavel V. Bondarenko, Zhongqi Zhang ⇑ Process and Product Development, Amgen, Thousand Oaks, CA 91320, USA article info Article history: Received 14 November 2010 Received in revised form 10 January 2011 Accepted 13 January 2011 Available online 19 January 2011 Keywords: Automation Protein Digestion Autosampler Mass spectrometry Monoclonal antibody Peptide mapping abstract A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation proto- cols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS sys- tem has great utility in preparing and analyzing multiple samples for protein characterization, identifi- cation, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control. Ó 2011 Elsevier Inc. All rights reserved. Due to their predictable properties, controlled functions, and long life in circulation, monoclonal antibodies (mAbs) 1 have be- come one of the most popular biotechnology medicines in develop- ment. Selection of a best monoclonal antibody drug candidate against a target of interest, followed by selection of a high-expres- sion clone, optimization of production and formulation parameters, and identity and quality control of mAbs and other therapeutic pro- teins, requires multiple peptide mapping analyses. Mass spectrometry (MS) has become the most important tool for structural characterization of mAbs and other therapeutic pro- teins [1]. With the development of automated high-performance liquid chromatography (HPLC) and MS instruments, sample prepa- ration and data analysis became the two bottlenecks on the way to automated, reproducible, and quantitative peptide mapping in the biotechnology industry [2]. Our efforts in data analysis were ad- dressed in a previous communication [3]. In the current article, we address the other bottleneck—sample preparation. Since the early days of biotechnology, it was revealed that mAbs are difficult to denature and digest due to the tight folding, particularly in the CH3 region [4]. It was noted that hydrophobic peptides containing the unique complementarity-determining regions (CDRs) tend to be underrepresented in the peptide maps due to either precipita- tion or absorption to the walls of sample containers [2]. In addition, sample preparation should not introduce a significant amount of artificial modifications to the protein [5,6] such as deamidation [7,8], pyroglutamate formation [2], hydrolysis of succinimide [9– 11], oxidation, disulfide scrambling, and transpeptidation [12,13]. These sample preparation challenges needs to be addressed to achieve high sequence coverage, high protein recovery, and quan- titative representation of all posttranslational modifications. Fortu- nately, peptide mapping of therapeutic proteins usually has the advantage of the availability of large amounts of the high-concen- tration proteins. Keeping these in mind, we developed an auto- mated digestion method that uses a relatively high concentration of a recombinant protein and multiple dilution steps to achieve denaturation, reduction/alkylation (if necessary), and enzymatic digestion of the protein. Previously, identification of proteomes of different organisms from two-dimensional gels led to the development of automated in-gel digestion [14,15]. The robots and protocols for automated proteolytic digestion became available commercially, and improvements in protocols of automated in-gel digestion continue to this day [16]. In addition, automated protein digestion was per- formed through online digestion using immobilized enzyme 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.01.019 ⇑ Corresponding author. Fax: +1 805 376 2354. E-mail address: zzhang@amgen.com (Z. Zhang). 1 Abbreviations used: mAb, monoclonal antibody; MS, mass spectrometry; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; GuHCl, guanidine–HCl; TFA, trifluoroacetic acid; DTT, dithiothreitol; TCEP, tris(2- carboxyethyl)phosphine; IAM, iodoacetamide; EDTA, ethylenediaminetetraacetic acid; IgG2, immunoglobulin 2. Analytical Biochemistry 411 (2011) 284–291 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio