An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2‐CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme‐independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc‐solid‐phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc‐SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C‐terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full‐length domain.

Indeed, the quality of a protein-based drug may be affected by the existence of multiple variants displaying altered biophysical and biochemical properties; such chemical diversity mainly arises from posttranslational modifications (PTMs), like glycosylation, and spontaneous degradation during the production and formulation processes. 8,9 To this regard, the most commonly encountered enzyme-independent modifications are methionine oxidation, [10][11][12] deamidation of asparagine coupled to succinimide (Snn), aspartate and isoaspartate (isoAsp) formation, [13][14][15][16] N-terminal glutamate or glutamine cyclization to pyroglutamate, [17][18][19] and arginine glycation or carbonylation. 20,21 Spontaneous degradation products due to pyroglutamate formation, methionine oxidation, and asparagine deamidation have been observed in therapeutic mAbs. 22 In particular, the IgG1 Fc domain contains two conserved and solvent-exposed methionines at positions 432 in the CH3 domain ( Figure 1) and 256 in the CH2 domain, which have been shown to be susceptible to oxidation upon manufacturing and storage. 11 Moreover, it has been demonstrated by surface plasmon resonance that oxidation of Met-432 and Met-256 impacts the binding affinity to the human neonatal Fc receptor (FcRn), 23 protein A, and protein G, 24 which has been attributed to the alteration of the secondary structure surrounding the residues involved in the binding. 25 Asparagine deamidation and related products (Asp, isoAsp, and Snn) have been detected in both the Fc and Fab regions of mAbs, causing alterations of their secondary structure, potency, and binding affinity, especially when occurring across the flexible complementarity-determining region, [26][27][28] thus having direct consequences on the Ab-mediated immune response. In the IgG1 Fc region, such PTM has been reported at Asn-319 within the sequence Leu-Asn-Gly-Lys in the CH2 domain, as well as at Asn-388, Asn-393, and Asn-438 within the CH3 domain [29][30][31] (Figure 1). The biological impact of deamidation has been studied in the Fab region of mAbs, [26][27][28] whereas its effect on the Fc region remains to date unclear. However, the results of an alanine-scan on human IgG1 have revealed that replacement of Asn-438 influences the binding affinity for FcRn. 32 Therefore, also the structural change caused by deamidation at Asn-438 might potentially affect the FcRn binding.
Thus, oxidation and deamidation events may be considered two of the most important sources of enzyme-independent PTMs in drugs based on mAbs and Fc-fusion proteins. Moreover, both changes may influence the Ig binding to FcRn, thus affecting the half-life of Igs and Fc-fusion proteins in the blood. [33][34][35] Although spontaneous PTMs are fortunately present only as minimal impurities, which may be hardly detectable even by highly sophisticated MS analysis, 36,37 nevertheless, the assessment of their structural and biological effects is of high significance. For this reason, it is often necessary to treat the protein chemically, in order to induce and investigate the desired PTM. For example, H 2 O 2 treatments are usually performed to increase the amount of oxidized methionine. This, however, results in multiple combinations of reduced and oxidized methionine residues. 11,23,38 In contrast, chemical protein synthesis (CPS) based on the native chemical ligation (NCL) of synthetic fragment precursors would, in principle, allow obtaining only one species at the time by replacing only the desired methionine with methionine sulfoxide. CPS has been successfully applied for small and medium-size proteins, [39][40][41][42][43] whereas other approaches have been developed to overcome the protein-size limitation, which rely on the NCL between synthetic and recombinant fragment precursors (socalled expressed protein ligation [44][45][46], or on the incorporation of unnatural amino acids (AAs) into a protein by genetic-code expansion. [47][48][49] At the light of the important role played by the CH3 domain in the binding of IgG molecules to FcRn 50,51 as well as in the dimerization process of minibodies, 4 we undertook the present work to assess the accessibility of this domain by chemical synthesis, which, in turn, would make variants containing, for example, oxidized methionine or D-amino acids at the desired position also accessible. In particular, we focused on the preparation of IgG1 CH3 fragment precursors for two possible NCL strategies that rely on threonine-based 52,53 and cysteine-based 43,54 chemoselective reactions (Scheme 1). The C-terminal fragment precursor (either 1, 4, or 6) contains oxidized methionine at position 432.
Problems encountered during the solid-phase peptide synthesis (SPPS) of the precursors and some solutions to them, as well as NCL attempts, are discussed below. Although the total chemical synthesis of the IgG1 Fc CH3 domain could not be achieved in this work, the conducted explorative study provides useful insights into the Fmoc-based SPPS of IgG1 Fc CH3 sequences, including C-terminally activated intermediates bearing the salicyladehyde ester (Sal) or a thioester.

| Methods
Analytical RP-HPLC was performed using a Thermo Fisher Scientific Dionex UltiMate 3000 UHPLC system (Germering, Germany) and either a Syncronics C-18 column (100 Å, 5 μm, 250 × 4.6 mm, Thermo Fisher Scientific) at a flow rate of 1.5 mL/min or a Nucleosil C-18 column (100 Å, 5 μm, 250 × 4 mm, Macherey-Nagel, Düren, Germany) at a flow rate of 1 mL/min. Unless specifically stated, the first one was used. The UV detection was set at 220 nm. The elution system was
The latter (0.04 g, 0.11 mmol) was dissolved in DMF, and the resulting solution was added to pre-swelled Rink amide MBHA resin (0.05 g, 0.022 mmol) together with HBTU (0.042 g, 0.11 mmol) and DIPEA   After 2.5 hours, the C-terminal Nbz peptide 1c was completely consumed, and no further changes were observed with respect to the previous control (Scheme 3C). Therefore, the reaction mixture was acidified, and the HPLC fraction containing the ligation product 1 was collected and lyophilized. The mass spectrum confirmed the successful separation of the desired ligation product 1 from all impurities present in the NCL mixture. However, an additional peak at slightly higher retention time appeared in the HPLC profile, whose identity could not be assessed by mass spectrometry. For the synthesis of the C-terminal Nbz peptide 3, we first functionalized the Rink amide MBHA resin (0.45 mmol/g) with mono-Fmoc-diaminobenzoic acid synthetized from commercially available 3,4-diaminobenzoic acid. The first residue, Fmoc-Thr(tBu)-OH, was coupled manually with HATU/DIPEA, followed by spectrophotometric determination of the Fmoc group, which confirmed the success of the coupling. The peptide chain was then elongated by automated SPPS with the HBTU/HOBt/DIPEA coupling protocol. After the assembly was completed, we attempted to convert the Dbz group into the Nbz group. However, the conversion was incomplete, probably due to the sterically hindered C-terminal Thr(tBu)-370. Thus, we decided to cleave the peptide from the resin in the Dbz form 3a, which is more stable than the Nbz form 3 but may be orthogonally converted into a benzotriazole moiety with NaNO 2 and then displaced by the thiol additive in the ligation buffer to afford the peptide thioester in situ. 73 The TFA-cleaved product 3a showed satisfactory homogeneity (~60%) (Scheme 5).   breakers like pseudoprolines and the use of low-loaded, highly swelling resins partially reduced the formation of aggregates, but this was not sufficient to obtain sequences larger than 44 residues, as also shown for the 58-residue long fragment 371 to 428 (5) (Figure 2).
Thus, the assembly route to obtain the IgG1 Fc CH3 sequence was Nbz on a peptide bearing a C-terminal leucine. 63 Unfortunately, in our case, the use of DMF did not solve the problem, and no Nbz peptide was formed (probably, because of the presence of the β-branched threonine). Thus, we activated the Dbz peptide 3a by converting it into an N-acyl-benzotriazole peptide by using NaNO 2 at pH~3, as previously shown by Liu and coworkers. 73 For the Acm cleavage from the C-terminal free acid of 8, we used the Pd-catalyzed reaction reported by Brik and coworkers. 92 Both reactions were successful, but, unfortunately, no ligation product was detected after 18-hour reaction. We did not try to change further the protocol, as the slow ligation rate of the C-terminal threonine is likely to prevent an efficient synthesis. An alternative would be to move the third ligation site between Gly-375 and Phe-376, which would be advantageous for the preparation of the C-terminal Nbz peptide precursor as well as for the ligation rate. However, β-mercapto-Phe must be used, whose synthesis and use in NCL have been previously reported. 93 Another way would be the stepwise synthesis of the segment 345 to 389, which would avoid a third ligation.
In conclusion, this explorative study has shed light on the behaviour of different fragments of the IgG1 Fc CH3 domain during the standard Fmoc-SPPS as well as NCL, which will be useful to plan future syntheses. Once completed the CPS of the CH3 domain, it will be necessary to test, if the synthetic bis(cysteinyl)-polypeptide chain can undergo oxidative folding and build the expected noncovalent homodimer.

FIGURE 2
Summary of the synthetic IgG1 Fc CH3 fragments of this study. All fragment precursors were obtained with homogeneity of~60% as crude products, except those colored in grey that were less homogeneous. The red/black ovals represent the ligation sites. The * represents Met(O)-432. On the top, the secondary structure (β-strands and helical turns) composition of the IgG1 Fc CH3 domain is shown (based on the crystal structure with PDB ID 1OQO)