Application of nSMOL coupled with LC‐MS bioanalysis for monitoring the Fc‐fusion biopharmaceuticals Etanercept and Abatacept in human serum

Abstract The principle of nano‐surface and molecular‐orientation limited (nSMOL) proteolysis has a unique characteristic Fab‐selective proteolysis for antibody bioanalysis that is independent of a variety of monoclonal antibodies by the binding antibody Fc via Protein A/G in a pore with 100 nm diameter and modified trypsin immobilization on the surface of nanoparticles with 200 nm diameter. Since minimizing peptide complexity and protease contamination while maintaining antibody sequence specificity enables a rapid and broad development of optimized methods for liquid chromatography‐mass spectrometry (LC‐MS) bioanalysis, the application of regulatory LC‐MS for monitoring antibody biopharmaceuticals is expected. nSMOL is theoretically anticipated to be applicable for representative Fc‐fusion biopharmaceuticals, because Protein A/G‐binding site Fc exists on the C‐terminus, and its functional domain is available to orient and interact with the reaction solution. In this report, we describe the validated LC‐MS bioanalysis for monitoring Ethanercept and Abatacept using nSMOL technology. The quantitation range of Ethanercept in human serum was from 0.195 to 100 μg/mL using the signature peptide VFCTK (aa.43‐47), and that of Abatacept was from 0.391 to 100 μg/mL using the signature peptide MHVAQPAVVLASSR (aa.1‐14). Both proteins fulfilled the guideline criteria for low‐molecular‐weight drug compounds. The results indicate that the clinical and therapeutic monitoring for antibody and Fc‐fusion biopharmaceuticals are adequately applicable using nSMOL proteolysis coupled with LC‐MS bioanalysis.


| INTRODUCTION
Tumor necrosis factor α (TNFα) is one of the proinflammatory cytokines that plays an important role in the pathogenetic signals in sepsis and several inflammatory diseases, especially playing the role of a mediator of systemic inflammation. 1,2 Recent studies focusing on TNFα signaling have indicated that endogenous TNFα is a key mediator in specific inflammatory responses. 3,4 The neutralizing TNF antagonists or monoclonal antibodies to TNF have exhibited clinically effective outcomes for many immune-mediated inflammatory diseases (IMIDs) such as rheumatoid arthritis (RA), 5,6 inflammatory bowel disease (IBD), 7 psoriatic arthritis (PsA), 5 vasculitis, 8 ankylosing spondylitis (AS), 9 and juvenile chronic arthritis (JCA). 10 Infliximab, Adalimumab, and Etanercept have been shown to have good therapeutic outcomes in various clinical trials since the first biopharmaceutical agents for IMID were launched in 1998. 11 TNF blockade strategy is an extremely important option for first-line biopharmaceuticals. Infliximab and Adalimumab have immunoglobulin G (IgG)based chimeric and human antibody structures, and Etanercept is a dimeric fusion protein consisting of the extracellular domain of the human p75 TNFα receptor II (TNFR) and Fc domain. 12,13 Discovered in 2005, Abatacept belongs to a new class of IMID therapeutic agents, which excludes the neutralizing proinflammatory cytokines. [14][15][16][17] Abatacept is a fusion protein comprising the extracellular domain of the inhibitory molecules cytotoxic T-lymphocyte antigen 4 (CTLA-4) and Fc of human IgG. CTLA4 has a higher affinity to CD80 and CD86 on antigen-presenting cells than to CD28 on T cells. Abatacept selectively regulates the CD80/86 costimulatory signals for T-cell activation, and is efficient for suppressive inflammatory observation. 18,19 All biologics and Fc-fusion molecules are proteins, and therefore, intrinsically possess immunogenic potentials for B-cell and T-cell epitopes. Since therapy against IMIDs requires a long-term and repeated administration for clinical efficacy, there is a constant potential to produce antidrug antibodies (ADA). [20][21][22] The existence of ADAs in patient circulation is considered to be one of the possibilities for the decreased levels of protein pharmaceuticals in blood, the loss of efficacy, or drug-related adverse events. Therefore, monitoring for therapeutic protein pharmaceuticals is essential for good clinical signatures. 23 In order to adequately identify loss of clinical responses, determine dosage increase, or switch to next agents, it is important to obtain precise blood level information by therapeutic drug monitoring. Moreover, it is also known that the blood levels of drugs are influenced by the coexistence of ADAs depending on the analytical methods; 24 hence, the advances in the universal monitoring technology are required. 25,26 Bioanalysis technology by LC-MS has essential issues to overcome. LC-MS has two main technologies such as column chromatography and mass spectrometry. In order to maintain the highresolution of column separation and rapid repeated analysis in LC unit, excessive sample injection should be avoided. And in MS unit, to maintain quantitative ionization, it is necessary to avoid the ionization suppression effect as much as possible and maintain an appropriate ESI interface environment. Our nano-surface and molecular-orientation limited (nSMOL) proteolysis principle is the sole LC-MS bioanalysis technology ( Figure 1). Briefly, protein Fc domain is first collected via Protein A/G resin with the pore diameter of 100 nm from biological samples, so that the opposite site like Fab or fused domain will be oriented to the reaction solution in the pore.
And second, modified trypsin immobilized on the surface of nanoparticles with the diameter of 200 nm is reacted in this mixture of Protein A/G resin and nanoparticles. In this solid-solid reaction field, trypsin access to the substrate is physicochemically limited. Therefore, proteolysis reaction is selectively and effectively performed on the orienting domain to the solution like the complementarity-determining region (CDR) in IgG molecules, without a large excess of tryptic peptide matrix and extra protease contamination. 27 The Fc-fusion protein biopharmaceuticals described above have a human Fc F I G U R E 1 Schematic view of nSMOL reaction principle domain on the C-terminus. Therefore, nSMOL bioanalysis is theoretically applicable to Fc-fusion protein. In this report, we describe the development of validated LC-MS bioanalysis for monitoring Etanercept and Abatacept levels in human serum using nSMOL approach.

| The content determination of N-terminal peptide heterogeneity in Abatacept
In order to determine the ratio of the N-terminal peptide content of Abatacept, MRM optimization was performed using three synthetic candidate peptides MHVAQPAVVLASSR, AMHVAQPAVVLASSR, and MAMHVAQPAVVLASSR (summarized in Table 3). Information about each Abatacept peptide was obtained from DrugBank, Review report from Pharmaceuticals and Medical Devices Agency (PMDA) Japan, and predicted from KEGG Drug, respectively. And then, nSMOL quantitation of Abatacept (10 and 100 μg/mL) was carried out in PBS buffer and in human serum. Each MRM intensity was normalized by 10 fmol of P14R ISTD intensity (Table 4).

| Preparation of sample for validation by nSMOL proteolysis
In the current study, we performed a bioanalytical validation of Etanercept or Abatacept in human serum using the nSMOL method as described in our previous report with a minor improvement. The nSMOL proteolysis coupled with the LC-MS/MS method was  CTLA-4, respectively. These molecules have a highly complexed structure by disulfide formation. Moreover, these receptors express their physiological functions by dimerization or trimerization. 28,29 Additionally, these Fc-fusion proteins have no hinge-like portions between the Fc and fused domains. Therefore, we analyzed the proteins was successfully proceeded independent of the difference of structural chemistry and molecular dynamics from IgG family.

| The limitation regarding the selection of signature peptide for Fc-fusion biopharmaceuticals
We have previously defined that the signature peptide of antibody drugs should be selected as follows: peptides with from 8 to 15 amino acid residues, with no cysteine residue, with no missed cleavage in the tryptic reaction, not in the vicinity of the disulfide bonding, with specific sequences containing CDR sequences against endogenous IgGs, and with no N-and C-terminus sequences because of amino acid heterogeneity on terminal fragment. 30 However, for performing the Etanercept and Abatacept assay development using LC-MS, we could not select the signature peptide according to our criteria because the selection of signature peptide was markedly limited by the complexed disulfide structure and low content of lysine and arginine residues aligned by ClustalW analysis shown in Figure 2. As a consequence, there was no choice but to select the cysteine-containing peptide of Etanercept and N-terminus peptide of Abatacept as candidate signature peptides for the nSMOL bioanalysis, respectively ( Figure 3).

| The stability of cysteine-containing peptide as a signature peptide of Etanercept in LC-MS bioanalysis
The representative oxidative product of the cysteine-containing peptide is formed by the dimerization via disulfide bridge formation. Therefore, we verified the ratio of the reduced and oxidative form, reversibility, stability, and oxidative tolerance in nSMOL reaction using a H 2 O 2 -treated dimerization peptide VFCTK. These results are summarized in  There are several reports on the N-terminal sequence of Abatacept. 31 We investigated the sequence information from two databases (DrugBank and KEGG Drug) and one document (PMDA) in  Table 4 indicates that the most frequent peptide was MHVAQPAVVLASSR at about a 60% content, and this ratio was constant and not degraded during the nSMOL reaction condition in PBS buffer and serum. Therefore, we have decided to use the peptide MHVAQPAVVLASSR for further validation assay.