Meiqi Yi,Jian Sun,Hanzi Sun,Yifei Wang,Shan Hou,Beibei Jiang,Yuanyuan Xie,Ruyue Ji,Liu Xue,Xiao Ding,Xiaomin Song,April Xu,Chichi Huang,Quan Quan,Jing Song
Identification and characterization of an unexpected isomerization motif in CDRH2 that affects antibody activity MABS
Binding activity,CDR,DHK motif,isomerization hot spot,PK
ABSTRACT Aspartic acid (Asp) isomerization is a spontaneous non-enzymatic post-translation modification causing a change in the structure of the protein backbone, which is commonly observed in therapeutic antibodies during manufacturing and storage. The Asps in Asp–Gly (DG), Asp–Ser (DS), and Asp–Thr (DT) motifs in the structurally flexible regions, such as complementarity-determining regions (CDRs) in antibodies, are often found to have high rate of isomerization, and they are considered “hot spots” in antibodies. In contrast, the Asp-His (DH) motif is usually considered a silent spot with low isomerization propensity. However, in monoclonal antibody mAb-a, the isomerization rate of an Asp residue, Asp55, in the aspartic acid-histidine-lysine (DHK) motif present in CDRH2 was found to be unexpectedly high. By determining the conformation of DHK motif in the crystal structure of mAb-a, we found that the Cgamma of the Asp side chain carbonyl group and the back bone amide nitrogen of successor His were in proximal contact, which facilitates the formation of succinimide intermediate, and the +2 Lys played an important role in stabilizing such conformation. The contributing roles of the His and Lys residues in DHK motif were also verified using a series of synthetic peptides. This study identified a novel Asp isomerization hot spot, DHK, and the structural-based molecular mechanism was revealed. When 20% Asp55 isomerization in this DHK motif occurred in mAb-a, antigen binding activity reduced to 54%, but the pharmacokinetics in rat was not affected significantly. Although Asp isomerization of DHK motif in CDR does not appear to have a negative impact on PK, DHK motifs in the CDRs of antibody therapeutics should be removed, considering the high propensity of isomerization and impact on antibody activity and stability.
Identification and characterization of an unexpected isomerization motif in CDRH2 that affects antibody activity MABS

Aspartic acid (Asp) isomerization is a spontaneous non-enzymatic post-translation modification causing a change in the structure of the protein backbone, which is commonly observed in therapeutic antibodies during manufacturing and storage. The Asps in Asp–Gly (DG), Asp–Ser (DS), and Asp–Thr (DT) motifs in the structurally flexible regions, such as complementarity-determining regions (CDRs) in antibodies, are often found to have high rate of isomerization, and they are considered “hot spots” in antibodies. In contrast, the Asp-His (DH) motif is usually considered a silent spot with low isomerization propensity. However, in monoclonal antibody mAb-a, the isomerization rate of an Asp residue, Asp55, in the aspartic acid-histidine-lysine (DHK) motif present in CDRH2 was found to be unexpectedly high. By determining the conformation of DHK motif in the crystal structure of mAb-a, we found that the Cgamma of the Asp side chain carbonyl group and the back bone amide nitrogen of successor His were in proximal contact, which facilitates the formation of succinimide intermediate, and the +2 Lys played an important role in stabilizing such conformation. The contributing roles of the His and Lys residues in DHK motif were also verified using a series of synthetic peptides. This study identified a novel Asp isomerization hot spot, DHK, and the structural-based molecular mechanism was revealed. When 20% Asp55 isomerization in this DHK motif occurred in mAb-a, antigen binding activity reduced to 54%, but the pharmacokinetics in rat was not affected significantly. Although Asp isomerization of DHK motif in CDR does not appear to have a negative impact on PK, DHK motifs in the CDRs of antibody therapeutics should be removed, considering the high propensity of isomerization and impact on antibody activity and stability.

Therapeutic monoclonal antibodies (mAbs), in particular IgGs, are now an important class of medicines that have applications in various therapeutic areas, including cancer, inflammatory disease, organ transplantation, cardiovascular disease, pathogenic infection, respiratory disease, and ophthalmological disease, due to several desirable features, such as high specificity, high potency, good safety and long half-life. In the folded state, IgG contains three relatively independent fragments, two identical antigen binding fragments (Fabs) and a crystallizable fragment (Fc), linked by a hinge region. Each Fab has six complementarity-determining regions (CDRs), including three CDRLs from light chain and three CDRHs from heavy chain, that in the structure form the antigen binding site. The formation of antigen-antibody complex is mostly governed by electrostatic and hydrophobic interactions between amino acid residues of CDRs and target epitopes. Therapeutic mAbs degrade via multiple pathways during expression, purification, formulation, storage, and delivery. Post-translational modifications (PTMs) or chemical modifications of CDR amino acid residues may have negative effects on antigen binding, leading to a decrease in potency, and therefore characterization of chemical modifications is an important part of evaluating the stability of therapeutic mAbs. Asp isomerization is one of the major chemical modifications of proteins under typical processing formulation and storage conditions. In mildly acidic buffers, aspartic acid (Asp) residues can form a cyclic imide intermediate, succinimide (Asu), via nucleophilic attack of the carbonyl group on the Asp side chain by the amide nitrogen on the backbone. The Asu loop is unstable under alkaline pH conditions and can be rapidly hydrolyzed into Asp and isoaspartic acid (isoAsp) residues (Figure 1). Previous studies have reported a 1:3 ratio of Asp to isoAsp from the succinate intermediate. The rate of isomerization reaction depends on various factors, including pH, temperature, primary structure, higher order structure, ionic strength, and other buffer conditions. Generally, antibody isomerization is more readily observed after incubation at elevated temperature and low pH. The formation of isoAsp may lead to an immune response and the loss of biological activity. Previous studies with small flexible peptides have shown that the reactivity of the side chains of amino acids next to Asp has the greatest impact on the rate of isomerization. This residue affects isomerization through steric, electrostatic, or catalytic interactions, and Gly, Ser, and Thr next to the Asp generally increase the rate of Asp isomerization. In addition, the conformational flexibility around Asp is also an important factor affecting isomerization propensity. The CDRs of antibodies bind to the therapeutic target and are mostly flexible in structure and solvent exposed. The observation of Asp isomerization in the CDRs of antibodies has become increasingly common. Irudayanathan et al. reported that backbone secondary structure, side-chain rotamer conformation and solvent accessibility were found to be key molecular indicators of Asp isomerization in 131 clinical-stage therapeutic antibodies. The observation of 131 clinical-stage therapeutic antibodies showed that Asp–Gly, Asp–Ser, and Asp–Thr in the CDRs were often found to have high rates of Asp isomerization and the obligate Asu intermediate is not likely to occur when the n + 1 residue is bulky. The observations of isomerization at the DH motifs are expectedly rare, with the DH motif usually considered a silent spot with low isomerization propensity. In this study, we found that the Asp55 in CDRH2 of an antibody, mAb-a, underwent very fast isomerization, and that this modification affected the target binding activity of the antibody. However, isomerization of Asp55 did not affect the pharmacokinetics (PK) of mAb-a in rat. Further characterization of the isomerization of Asp55 of mAb-a revealed a novel isomerization motif, DHK, and the structural roles that His and Lys of this motif played in increasing Asp isomerization propensity.

Samples of mAb-a stored under mildly acidic pH conditions (5.0 and 6.0) were analyzed by cation ion exchange (CIEX) chromatography, and showed charge variant heterogeneity (Figure 2A). The chromatogram consisted of three fractions designated M, B1, and B2, of which both B1 and B2 were the basic peaks of mAb-a, indicating charge heterogeneity of mAb-a. As the incubation time increased, the ratio of the basic peaks (B1 and B2) of mAb-a also increased. After incubation at pH 5.0 and 25∘C for only 5 days, the level of the base peaks increased dramatically from 27.1% to 72.9%, and the level of the main peaks decreased from 57.0% to 21.4%. Moreover, after incubation at pH 6.0 for 5 days, the basic peaks of mAb-a also increased significantly to approximately 45.8% (Table 1). Surprisingly, under neutral conditions (pH 7.0) and extremely acidic conditions (pH 3.5), the base peaks of mAb-a did not change significantly in relative intensity. In summary, the CIEX chromatograms indicated that mAb-a underwent a rapid change over a narrow range of pH of around pH 5.0, specifically under slightly acidic conditions. The main peak and basic peaks (B1 and B2) of pH 5, 5 days pre-incubated sample were individually collected and analyzed through intact mass spectrometry (MS). The results revealed that the B1 peak had a molecular mass 18 Da lower than that of the main peak, while B2 peak had a 36 Da lower mass. These findings indicate that succinimide, rather than IsoD, was the primary isomerization product following mild acidic treatment (Figure 2B). Moreover, the B1 and B2 peaks were attributed to species with a single heavy chain and both heavy chains containing succinimide modifications, respectively.

To identify the major changes in mAb-a at low pH, mAb-a was incubated under different pH conditions (pH 5.0, 6.0, and 7.0) at room temperature, and tryptic peptide mapping was used to measure the chemical modification levels. We found that under mildly acidic pH conditions, a high proportion of succinimide was formed from Asp55 in CDRH2 of mAb-a. As mentioned above, succinimide is the intermediate of aspartic acid isomerization. Figure 3 shows the combined extracted ion chromatograms of tryptic peptide P1 containing aspartic acid, isoaspartic acid, or succinimide extracted from mAb-a incubated under the indicated pHs. P1 contains two aspartic acids (Asp52 and Asp55) that can potentially undergo isomerization. In the chromatogram of peptide P1 containing the succinimide residue, there were multiple peaks corresponding to the calculated b and y ions, confirming the presence of the succinimide residue at the position of Asp55 (Figure 3). At the retention time of 30.54 min, the observed peak corresponded to m/z of 699.08 (3+), which matched the calculated molecular weight, and this peak was assigned to the peptide with the original aspartic acid residue. The peak with the retention time of 31.24 min had the same molecular weight as the main peak, and MS/MS indicated that the two peaks had the “same” amino acid sequence, indicating that the later peak of 31.24 min could correspond to the same peptide containing the isoaspartic acid residue. The calculated m/z of the peak at 29.75 min was 694.58, which was 18 Da below the theoretical molecular weight and thus corresponded to a peptide with the succinimide residue. Because Asp and isoAsp were indistinguishable in the MS/MS spectra, synthetic peptide P1 and its isoAsp isomer, iso-P1, were generated for verification of peak identification. The synthetic peptide P1 was eluted at 30.54 min, while iso-P1 had the same retention time at 31.24 min as each later eluting peak, which confirmed the later eluting peak at 31.24 min was isomerized peptide iso-P1 (data not shown). The intensity of peaks eluting later (31.24 min) or earlier (29.75 min) increased under acidic (pH 5.0 or 6.0) incubation conditions. As shown in Table 1, after mAb-a was incubated for 5 days at pH 5.0, the level of the isoAsp55 reached 26.5% and the level of succinimide reached 16.8%, indicating that modification at this Asp55 site was the major cause of basic peaks increase in the CIEX chromatograms. At the intact mass level, our results revealed that the primary isomerization product of mAb-a induced at acidic pH was succinimide. However, during peptide mapping analysis, the experimental conditions could disrupt the succinimide and convert it into either isoAsp or Asp. Therefore, the high proportion of isoAsp observed in the peptide mapping analysis was due to the degradation of succinimide over the analytical experiment process.

mAb-a was pre-incubated in different pH conditions (pH 5.0, 6.0, and 7.0) to determine the effect of isomerization on antibody binding potency. CHO-K1 cells that overexpressed the target antigen were used to determine the relative binding activity of mAb-a using fluorescence-activated cell sorting (FACS). As described in the methods section, we normalized the binding activity (measured by EC50) of untreated mAb-a as 100%. The relative binding activity of the treated sample was estimated by normalizing it against untreated mAb-a. The binding potency showed a clear trend of decrease over pre-treatment incubation under acidic pHs (Figure 4A). As previously discussed, succinimide was found to be the primary product of mAb-a isomerization under mild acidic pH conditions. However, during the liquid chromatography (LC)-MS peptide mapping analysis, a significant portion of succinimide was converted to isoAsp as well as Asp. Therefore, the sum of %isoD and %succinimide was used to assess the correlation between Asp55 isomerization and the relative binding potency of mAb-a. There was a good correlation between mAb-a potency and Asp isomerization; specifically, target cell binding activity decreased when the amount of isomeric Asp increased (Figure 4B). Size-exclusion chromatography (SEC) and capillary gel electrophoresis (CGE) testing results showed minimal changes in mAb-a aggregation and fragmentation levels for pH 5.0 and 6.0 incubated samples (data not shown). The results showed the isomerization of this Asp55 residue led to decreased antibody activity, and suggested Asp55 in CDRH2 was directly involved in target binding.

To determine the rate of Asp55 isomerization in vivo, Sprague Dawley (SD) rats were injected with a dose of 10 mg/kg mAb-a pre-treated under indicated pH conditions for 5 days. mAb-a in rat plasma sample was captured and analyzed using the immunoprecipitation (IP)-LC-MS/MS method (Figure 5). A portion of the captured antibody was digested by trypsin for PTM analysis using LC-MS/MS and another portion was used for antibody quantitation analysis. Figure 6 shows the relationship between isoAsp% and time after injection of different mAb-a in rats. After 7 days in vivo, the level of the isoAsp55 of mAb-a pre-treated at pH 5.0 (mAb-a-pH5), 6.0 (mAb-a-pH6), and 7.0 (mAb-a-pH5) increased by approximately 15%, 14%, and 14%, respectively. Although the plasma was considered to maintain the neutral pH (pH7.3-pH7.5) in vivo, it did not prevent the isomerization of Asp55 in mAb-a, indicating that mAb-a was unstable in vivo irrespective of the initial state of mAb-a Asp55, that is, pre-treatment of mAb-a under different pH conditions did not affect Asp55 isomerization rate in vivo. The succinimide intermediate was not detected by IP-LC-MS/MS. This was expected because the IP-LC-MS/MS experiment was performed in a slightly alkaline (pH 7.5) solution for optimal binding affinity. Under this pH condition, the succinimide intermediate could be rapidly hydrolyzed to form isoaspartic acid and aspartic acid, and thus isoaspartic acid is the major modified species detected.

MAb-a was pre-incubated under different pH conditions and then intravenously (IV) administered to rats. An ELISA-based quantitative assay was used to determine the mean concentration–time curves of mAb-a, as shown in Figure 7. The results showed that mAb-a samples pre-incubated under different pH conditions appeared to have similar exposure over time profiles in rat. The parametric area under the curve from days 0 to 7 (AUC0–7), was highly comparable among the three groups (Table 2). The Cl of mAb-a-pH5 was 0.650 mL/h/kg, while the Cl of mAb-a-pH6 and mAb-a-pH7 was 0.628 and 0.632 mL/h/kg, respectively, both of which met the criteria for bioequivalence. Other PK parameters such as Cmax and half-life were also highly comparable. Thus, mAb-a containing different starting levels of Asp55 isomerization exhibited similar PK profiles in rat. To verify this observation, we established a standard curve using LC-MS/MS data and quantified the concentration of mAb-a in the plasma by the IP-LC-MS/MS method. The IP-LC MS/MS results were consistent with the ELISA results and provided orthogonal confirmation of the ELISA determination of antibody level in plasma (data not shown). Overall, antibody concentration over time profiles was similar among mAb-a with different Asp55 isomerization levels after a single IV dose of 10 mg/kg in SD-rats. There were no significant differences in all PK parameters among the mAb-a isomerization variants. The results showed that the isomerization at Asp55 appeared to have no significant impact on mAb-a PK.

To investigate the molecular mechanism of high isomerization propensity of this Asp55 residue, the relative contribution of the primary sequence and structural conformation to this Asp isomerization was determined. A synthetic peptide P1 with the sequence corresponding to that adjoining Asp55 of mAb-a was incubated at pH 5.0, 6.0, and 7.0, with all samples at room temperature, for 7 days. The isomerization products were injected onto the same C18 column and assayed by RP-HPLC using the same gradient as in mAb-a tryptic peptide mapping. The results were consistent with those of previous protein-level studies, showing a typical profile of isomerization peptide and with the amount of isomerized product increasing over time (Figure 8). The isomerization level rose over seven days by 17%, 12%, and 2% at pH 5.0, 6.0, and 7.0, respectively. This result demonstrates that the synthetic peptide containing the primary sequence motif exhibited a similar isomerization rate as mAb-a, suggesting that the primary sequence may be the determining factor for the observed isomerization. To evaluate the relative roles of each adjacent residues surrounding Asp in the primary sequence motif, TSDHKT, we synthesized different mutant variants of peptide P1 to obtain peptides M1, M2, M3, M4, and M5, which were subjected to different pH conditions as above. The results are shown in Figure 9. When H (position +1) or K (position +2) was mutated to alanine (A), the rate of Asp isomerization was significantly reduced and increased by only 5% within 7 days at pH 5.0. In contrast, the alanine mutations at the other 3 adjacent positions (positions −2, −1, and +3) appeared to have no significant impact on the isomerization rates. These results show that the DHK motif in the synthetic peptide is sufficient to promote Asp isomerization, confirming that the primary motif, DHK, plays a crucial role in Asp reactivity. As it was unexpected based on literature knowledge that the His at position +1 and Lys at position +2 played important roles in Asp isomerization propensity, we hypothesized that this high propensity might be due to some structural conformation. The X-ray crystal structure of mAb-a Fab fragment was determined at 1.61 angstrom resolution (PDB:8JBJ). From this structure, Asp55 of mAb-a is located in a loosely ordered structure (Figure 10A). It is known that the successor residue of a reactive Asp residue had a greater effect on the reaction rates than the residues on the N-terminal. The reactivity of Asp may be affected by the structure of its successor residue, including steric hindrance, residue flexibility, and ionization state. Studies focusing on the primary sequence have suggested that the DH motif is not a hot spot for isomerization. As shown in Figure 10B, the side chain of His56 forms two hydrogen bonds with its own backbone carbonyl and backbone nitrogen of Arg72. These two hydrogen bonds stabilize the conformation that exposes the backbone amide nitrogen of His56. In addition, the salt bridge between the side chain of Lys57 and Asp55 further stabilize the ring-like conformation and narrow the distance between Cgamma of Asp55 and backbone nitrogen of His56. Therefore, the special structure of His56 and Lys57 contribute to the high rate of Asp isomerization. Moreover, synthetic DHK-containing peptide showed a similar conformation to the antibody structure through structural simulations, which were consistent with the high rate of Asp isomerization. Taken together, both His56 and Lys57 made a substantial contribution to the Asp isomerization observed in both antibody and DHK-containing peptide, indicating the DHK motif leads to a high propensity of Asp isomerization.

In this study, we found that isomerization of mAb-a Asp55, which is not in a conventional isomerization hot spot, occurred at a high rate and changed the basic peak levels of mAb-a in the CIEX analyses. In vivo experimental results showed that mAb-a Asp55 was also unstable in rat plasma at neutral pH, and the level of isomerized Asp55 increased 7 days post dose in rat. The Asp55 was located in the CDRH2 region of mAb-a, and we found that Asp55 isomerization affected the binding between the antibody and the target, thereby reducing the potency. However, Asp55 isomerization did not significantly affect the PK profile of mAb-a in rat, probably owing to the overall conformational stability of Asp55 isomerized mAb-a variant. To determine the mechanism of the high rate of Asp55 isomerization in the CDR, we further analyzed the adjacent primary sequence and local structural features from the crystal structure of the mAb-a Fab. We found that mutation of the DHK motif, that is, replacing histidine or lysine with alanine, dramatically slowed down Asp isomerization. From detailed structural analyses, the DHK motif is in the flexible β-turn, which is completely exposed and close in space. The +2 Lys residue forms a direct salt bridge with the side chain of Asp, which further reduces the distance between carbon atom of carboxylic acid side chain of Asp and backbone amide nitrogen of + 1 His. These effects indicate how the DHK motif in such a conformation promotes the isomerization of Asp. DHK motif is an unexpected hot spot that undergoes fast isomerization, which is a part of the critical quality attributes of the therapeutic antibody mAb-a. The identification of this DHK motif expands our understanding of the mechanism and kinetics associated with the aspartic acid isomerization reaction. It also provides atomic level insight into a favorable structure for the reaction. The newly identified DHK isomerization motif and structural insight of Asp isomerization may facilitate a better molecular design in the discovery stage of therapeutic biologics to minimize Asp isomerization risk.

MAb-a and its recognized Antigen-a were produced at BeiGene. The specific peptides were synthesized at the BankPeptide Co. (China). Dynabeads™ M-280 Streptavidin and EZ-Link™ NHS-biotin (Thermo Scientific) were used for IP-LC-MS/MS. Modified trypsin was purchased from Promega and IdeS was purchased from FabRICATOR. Other chemical reagents used in experiments were from Sigma. MAb-a was incubated in 20 mM NaAc buffer (pH5.0), 20 mM Histidine-HCl buffer (pH 6.0), and 20 mM Tris-HCl buffer (pH7.0) at room temperature for 2 h, 6 h, 24 h, 48 h and 120 h to obtain mAb-a-pH5, mAb-a-pH6, mAb-a-pH7 samples.

Cation-exchange chromatography was performed by salt gradient separation on a MabPac SCX-10 (Thermo Fisher Scientific) column using an Agilent 1200 HPLC system. The elution system had a gradient change from 0% to 50% B in 20 min at a flow rate of 1 ml/min, while mobile phase A was 20 mM sodium phosphate, pH 6.5, and mobile phase B was 20 mM sodium phosphate, 0.2 M NaCl, pH 6.5. The column temperature was kept at 37°C and UV absorbance was measured at 280 nm with Agilent G4212B Diode Array Detector. Data evaluation was performed via the evaluation function of chromatography software 32Karat. Below peak (main peak) or peak groups (acidic peak group, basic peak group) were identified for samples or standards.

MAb-a samples were diluted to 1 mg/mL, and then serially diluted in 96-well V-bottom plates at 1:4 ratio to a final concentration to 96-well plate. The concentrations of 10 points-serial dilution are as follows: 30 μg/mL, 7.5 μg/mL, 1.875 μg/mL, 0.46875 μg/mL, 0.117188 μg/mL, 0.029297 μg/mL, 0.007324 μg/mL, 0.001831 μg/mL, 0.000458 μg/mL, and 0.000114 μg/mL. 5 × 10 cells/well were resuspended and distributed in 200 μL of FACS buffer to new V-bottom 96-well plates, and blocked for 20 min. After blocking, the cells were added with 100 μL/well pre-diluted mAb-a samples, incubated at 4°C for 1 h, and washed by FACS buffer three times. After that, cells were added with Goat IgG F(ab’)2 anti-human IgG [F(ab’)2 specific]-488 (Jackson, 109-546-097, 1:200) as the secondary antibody, incubated at 4°C for 0.5 h, and washed by FACS buffer thrice. Following by the fixation with 1% paraformaldehyde, the cells were subjected to flow cytometry with Guava easyCyte 6HT Flow Cytometer (Millipore, USA).

2.4 mg mAb-a samples were denatured with 8 M GuHCl in 200 mM Histidine-HCl solution (pH 6.0) to obtain 120 μL, 2 mg/mL samples and then slightly vortex for a short time. 1.2 µL of 1.0 M dithiothreitol solution was added followed by incubation at 56°C for 1 h for reduction. After addition of 2.4 µL of the freshly prepared 1 M iodoacetamide solution, the samples were incubated at room temperature in dark for 30 min. MAb-a were then buffer-exchanged into 120 μL of 20 mM Histidine-HCl solution by Zeba Spin desalting column (Thermo Scientific, 0.5 mL Ref# 89882) following the manufacturer’s protocol. After desalting treatment, the concentration of mAb-a was determined by using Nanodrop with the UV absorption at 280 nm to confirm the loss of desalting step. Before digestion, samples were diluted with 20 mM, pH 6.0 Histidine-HCl buffer at a final concentration of 1.0 mg/mL, and tryptic digestion was performed at 37°C for 30 min using an enzyme/protein weight ratio of 1/20. Formic acid (FA) solution is added to sample to stop the digestion (final concentration 1% FA). After centrifugation, 5 µg sample in supernatant was injected into LC/MS. Digested samples can be stored at −80°C up to 30 days until LC-MS analysis.

The synthetic peptides were incubated in 20 mM NaAc buffer (pH5.0), 20 mM Histidine-HCl buffer (pH 6.0), and 20 mM Tris-HCl buffer (pH7.0), respectively, at room temperature for 2 h, 6 h, 24 h, 48 h, 72 h, 120 h and 168 h. The incubated products were separated using a CSH-C18, 2.1 × 100 mm, 1.7 µm column (Waters) at 65°C with a flow rate of 0.2 ml/min. The chromatographic condition was the same as that in the PTM identification method.

Male SD-rats were dosed at 10 mg/kg (mAb-a-pH5, mAb-a-pH6, mAb-a-pH7) during a 5-min intravenous infusion for three replicates. Blood from rats was collected via a vascular access port at 5 min, 2, 6, 24, 48, 120, and 168 h, and processed for plasma. The plasma samples were stored at −80°C until IP-LC-MS/MS and ELISA analysis. All procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC).

Antigen-a was biotinylated by mixing with biotin-NHS at w/w 10:1 ratio and incubating at room temperature for 1 h. Then extra Biotin-NHS was removed by Zeba desalting column, and biotinylated antigen-a was exchanged to HBS-EP buffer. 7 mL of 10 mg/mL M280 Streptavidin Dynabeads was mixed with 1 mg biotinylated antigen-a at room temperature for 1 h after 3 times wash with HBS – EP buffer (pH7.5). After incubation, Dynabeads were washed with 10 mL of HBS – EP buffer twice and Dynabeads binding with Antigen-a were resuspended in 7 mL HBS – EP buffer. Samples (100 μL of rat plasma) and 100 μL of Dynabeads were mixed in a 96-well plate, capped, and placed on a shaker for overnight at 4°C. The Dynabeads were washed three times with HBS – EP buffer and resuspended with 300 uL of HBS – EP buffer containing IdeS, incubated at 37°C for 1 h with gentle shaking to remove Fc fragments. The Dynabeads were washed with HBS – EP buffer twice, followed by 500 uL of water twice and 10% acetonitrile once. Following the bead washing steps, a 100 μL aliquot of 10% acetonitrile with 1% formic acid was added to each sample and vortexed for 15 min at room temperature. The supernatant was the final product containing mAb-a Fab enriched from rat plasma. The PTM analysis of mAb-a Fab was followed by the PTM identification assay by LC-MS/MS described above.

The microtiter plate (Nunc, Cat#442404) was immobilized with 1 µg/mL antigen-a (prepared in house, 3 mg/mL) overnight at 2–8ºC. On the next day, the plate was washed and blocked by 3% bovine serum albumin (BSA) in PBST (PBS with 0.05% Tween-20) for 2 hours at room temperature. At the same time, the range of the mAb-a standard calibration samples were prepared from 0 ng/mL to 120 ng/mL in 3% BSA/PBST. Plasma samples were prepared at 0.1% required dilution in 3% BSA/PBST buffer. The samples were added to the plate and incubated at room temperature for 2 h. The unbound material was washed away and the captured mAb-a was detected by incubating with labeled goat anti-human polyclonal antibody at room temperature for 1 h. After thoroughly washing, streptavidin−horseradish peroxidase (Sigma, Cat#5512) was added and incubated for another 1 h. Following addition of 3,3′,5,5′-tetramethylbenzidine (Sigma, Cat#T0440) to each well, the plates were incubated at room temperature for 10 minutes and add stop solution (0.32 M H2SO4) to stop the reaction. The concentration of mAb-a in plasma samples was calculated from electrochemiluminescence intensity as measured by SpectraMax Paradigm using a 4-parameter logistic calibration curve generated from mAb-a calibrators.

DNA sequences coding for the Fab of mAb-a were synthesized with codon-optimization in mammalian cells. The sequences of heavy and light chain of Fab were cloned into pMAX vector, respectively, with a C-terminal 6×HIS tag in heavy chain. The plasmids harboring heavy chain and light chain of mAb-a Fab were transiently co-transfected into HEK293 cells for protein expression. The supernatant containing secreted Fab was purified by TALON affinity resin (Clontech Laboratories), followed by further purification using a HiLoad 16/600 Superdex 75pg column (GE Healthcare Life Sciences). The purified protein of mAb-a Fab was concentrated to around 10 mg/ml in 20 mM Tris pH 8.0, 100 mM NaCl for initial crystallization screening. Crystals of mAb-a Fab were grown in 21% PEG3350, 0.4 M potassium formate. The crystals of mAb-a Fab cryoprotected with 21% PEG3350, 0.4 M potassium formate, 5% glycerol were flash frozen in liquid nitrogen. The X-ray diffraction data was collected at beamline BL45×U at Spring-8 synchrotron radiation facility (Hyogo, Japan).