Systematic characterization of lysine glucuronidation in a bispecific antibody

ABSTRACT

This study presents a systematic characterization of lysine glucuronidation that was revealed during the charge variant characterization of a bispecific antibody (bsAb). Site-specific quantitation by Glu-C/Asp-N peptide mapping suggested that glucuronidation occurred randomly across surface lysine residues. To understand the impact of glucuronidation on the structure and function of the bsAb, stressed samples with up to 84% total glucuronidation were generated and analyzed by a comprehensive panel of analytical methods. The results suggested that glucuronidation caused an acidic isoelectric point (pI) shift in the charge profile. However, it does not affect the higher-order structure or bioactivities of the bsAb, including antibody-dependent cell-mediated cytotoxicity, antigen binding, or Fc receptor interaction. To support routine process monitoring, a fit-for-purpose subunit mass method was developed and qualified for quantitation of glucuronidation, offering a higher-throughput alternative to peptide mapping for assessing process consistency and product comparability.

Introduction

Recombinant monoclonal and bispecific antibodies are known to have diverse structural variants due to post-translational modifications (PTMs), fragmentation, or aggregation.^1–4 Common modifications producing an acidic shift include deamidation, isomerization, glycation, N-terminal glutamine cyclization, and sialylation. In contrast, common modifications producing a basic shift include C-terminal lysine, C-terminal proline amidation, succinimide intermediate, N-terminal glutamic acid cyclization, and aggregation.^5,6 These charge variants can form at various stages of the antibody manufacturing process, and may affect potency, serum half-life, or product stability.^7,8 Capillary isoelectric focusing (cIEF) and ion exchange (IEX) chromatography are routinely used to monitor charge variant profiles and degradation products, and demonstrate comparability.^8,9

Among the acidic modifications, glycation is commonly observed, occurring from the nonenzymatic conjugation of glucose to surface lysine through the Maillard reaction.^10,11 This reaction occurs when a reducing sugar, such as glucose, reacts with a free amino group commonly found on lysine side chains.^10,12–14 Glycation is particularly prevalent during antibody cell culture production, as reducing sugars like glucose and galactose are added as sources of energy during upstream manufacturing for the Chinese hamster ovary (CHO) production system.^15–17 Additional sources of glycation have been reported due to heat-induced sucrose degradation, which occurs when a reducing sugar is present in the formulation buffer.^18–20

In contrast, glucuronidation, resulting from surface lysine modification through covalent conjugation of glucuronic acid, has only been sporadically reported in the literature.^5,21–23 Our systematic characterization, however, revealed that glucuronidation is commonly present in antibody acidic variants, albeit at low levels. Given the structural similarities between glucose and glucuronic acid, particularly the presence of reactive aldehyde groups in both molecules, it is plausible that glucuronic acid covalently attaches to surface lysine through mechanisms similar to glycation (Figure 1), but the exact root cause of glucuronidation remains under investigation. Both glycation and glucuronidation cause acidic pI shifts in the cIEF profile compared to the unmodified form.¹⁵

Figure 1.

Attachment of glucose and theoretical attachment of glucuronic acid to a lysine residue.

In this study, charge variants of a bispecific antibody, hereafter referred to as bsAb1, were thoroughly characterized. Charge isoforms were enriched through semi-preparative cation exchange (CEX)^8,24 chromatography using a pH gradient. CEX separates protein analytes based on their surface charge using solid-phase chromatography.^25,26 In contrast, cIEF separates proteins based on their pI during electrophoretic migration in a capillary.^27–32 The combination of these two complementary techniques enables a more comprehensive analysis of the charge variant profile of bsAb1, facilitating further characterization of the underlying compositions of individual charge variants.

Since both glucuronidation and glycation modify surface lysine residues and interfere with trypsin digestion, the enriched CEX fractions were digested using a combination of Glu-C/Asp-N enzymes, followed by liquid chromatography-mass spectrometry (LC-MS) peptide mapping with electron-transfer/higher-energy collision dissociation (EThcD)³³ fragmentation. This approach enabled the identification of site-specific glucuronidation within the protein sequence. While other common PTMs, such as deamidation, were also detected in the acidic region, they are beyond the scope of this work.

Although glucuronidation has been briefly mentioned in prior reports,^5,21–23 detailed structure/function characterization has not been explored. To the best of our knowledge, our study presents the first comprehensive structure/function assessment of lysine glucuronidation in an antibody product. Forced glucuronidation experiments were performed by exposing the bsAb1 to varying concentrations of glucuronic acid. The total glucuronidation levels were determined by intact mass and subunit mass analysis, while Glu-C/Asp-N peptide mapping was used to quantify this modification at the peptide level. These stressed samples were then used to assess the impact of glucuronidation on the cIEF charge profile and biological functions. Additionally, size exclusion chromatography (SEC), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS); reduced (R) and non-reduced (NR), circular dichroism (CD) spectroscopy, sedimentation velocity analytical ultracentrifugation (SV-AUC), and differential scanning calorimetry (DSC) were also performed on select glucuronic acid-stressed material to assess the impact on aggregation, fragmentation, and higher-order structures (supplemental data). This comprehensive approach enabled the investigation of the biochemical, biophysical, and functional impact of glucuronidation on this bispecific antibody.

Finally, we describe method qualification of a fit-for-purpose subunit mass analysis assay, designed to facilitate routine monitoring of glucuronidation and glycation. Our findings reveal that, due to limited mass resolution, the widely used intact mass analysis can yield misleading “total glycation” results when glucuronidation co-presents with glycation. In contrast, the subunit method effectively overcomes this limitation, enabling accurate quantitation of both modifications. This subunit method also provides higher throughput compared to peptide mapping. Overall, this method improves both the specificity and throughput of detecting and quantitating glucuronidation and glycation in protein samples.

Results

Charge heterogeneity characterization by cIEF analysis

Charge variants were effectively separated from the unmodified protein based on differences in their respective pI values using cIEF. Both glycation and glucuronidation typically lead to acidic pI shift due to the deprotonation of the epsilon-amino group on lysine residues during the early stages of the Maillard reaction.¹⁵ As depicted in the top panel of Figure 2, bsAb1 exhibits three acidic peaks, one main peak, and one prominent basic peak in the cIEF profile. The basic peak is associated with C-terminal proline amidation, commonly observed in antibody products.^34–36

Figure 2.

Comparison of cIEF and CEX separation of bsAb1. The top panel presents the cIEF electropherogram of bsAb1, which reveals a prominent main peak with acidic (left) and basic (right) regions bracketed by two pI markers. The bottom panel shows the corresponding CEX chromatogram, highlighting the acidic fractions (A1-A3), the main fraction, and the basic fractions (B1-B3). While comprehensive characterization was conducted on all fractions, this publication focuses on the data from acidic fractions and the main fraction.

Fractionation of the acidic species using CEX chromatography

To further characterize the acidic species, bsAb1 was fractionated at a semi-preparative scale using CEX chromatography. Fractions isolated from multiple injections were pooled, buffer exchanged, concentrated, and subsequently analyzed by cIEF, peptide mapping, and subunit mass analysis.

The acidic region of the CEX profile exhibited a pattern similar to that of cIEF in terms of the number of peaks and their relative intensities, as illustrated in the bottom panel of Figure 2. The acidic region was fractioned into three regions, designated as A1, A2, and A3. The main fraction contained the major species, while fractions B1-B3 represented the basic species. Although not a focus here, it is important to note that a more pronounced basic peak was observed in the CEX profile relative to that in cIEF. This discrepancy is primarily attributed to the presence of C-terminal lysine, which was enzymatically removed with carboxypeptidase B (CPB) prior to cIEF analysis. In contrast, the protein sample used for CEX fractionation was not treated with CPB to avoid any potential interference from residual enzymatic activity before downstream characterization of the enriched fractions.³⁷

The isolated CEX fractions were analyzed by cIEF, with the resulting electropherograms shown in Figure 3 and the corresponding quantitation data summarized in Table 1. CEX acidic fraction A1 was predominantly enriched in cIEF peaks 1, 2 and 3 (91.8% of the total peak area). CEX acidic fraction A2 was mainly enriched in cIEF peaks 2 and 3 (88.3% of the total peak area). CEX acidic fraction A3 was primarily enriched in cIEF peak 3 (77.0% of the total peak area), while the main peak was predominately enriched with cIEF peak 4 (86.4% of the total peak area). No basic peaks were detected in any of the isolated acidic or main fractions. These findings demonstrate effective enrichment of the CEX fractions as shown in the distinct electrophoretic profiles that highlight the effective separation of acidic components and the absence of basic peaks.

Figure 3.

cIEF electropherograms of the isolated CEX fraction.

Table 1.

Results from the cIEF analysis of the enriched CEX fractions. Results are presented as a percentage of the total peak area. Due to rounding, the sum may not equal 100%.

cIEF Charge Isoform	Unfractionated bsAb1	Fraction A1	Fraction A2	Fraction A3	Main Fraction
New Acidic Peak A	ND	7.1	ND	ND	ND
Peak 1 (Acidic)	2.1	33.8	10.0	1.8	ND
Peak 2 (Acidic)	7.8	49.2	35.3	15.6	ND
Peak 3 (Acidic)	24.0	8.8	53.0	77.0	13.4
Peak 4 (Main)	61.2	1.0	1.7	5.7	86.4
Peaks 5 & 6 (Basic)	4.9	ND	ND	ND	ND

ND = Not detected.

Site specific glucuronidation identified by peptide mapping analysis

To accurately identify glucuronidation sites at the peptide level, a bottom-up Glu-C/Asp-N peptide map with tandem EThcD fragmentation was utilized, following a similar approach previously used to characterize glycation sites.^15,38–41 Unlike harsher fragmentation techniques, such as CID or HCD, EThcD proved effective in preserving the peptide-glucuronyl bond, enabling accurate site-specific identification of glucuronidation.

Peptide mapping showed that the acidic fractions contain a mixture of glucuronidated bsAb1 at multiple lysine sites. Figure 4 illustrates the identification of glucuronidation sites by EThcD fragmentation. The Glu-C derived peptide, E.KHKVYACE.V, from the conserved light chain (κ) region was chosen as an example to illustrate glucuronidation site identification since it contains two lysine residues susceptible to glucuronidation: one at the n-terminal lysine and the other at the third amino acid position. Panel A shows the MS/MS spectrum of the native peptide. Panel B illustrates the glucuronyl addition on the N-terminal lysine, which exhibits a distinct +176.0321 m/z shift in the “c” series ions beginning with “c1” relative to the native peptide shown in Panel A. Similarly, Panel C shows the glucuronyl modification on the third lysine residue, evidenced by a mass shift of the “c” series ions beginning with “c3” relative to the native peptide presented in panel A.

Figure 4.

XIC and EThcD fragmentation of a native and glucuronyl modified κ light chain peptide E.KHKVYACE.V. (A) unmodified peptide. (B) N-terminal lysine glucuronidation, indicated by a +176.0321 Da mass shift in the c-ion series beginning at c1. (C) Glucuronidation at the third lysine residue, with c-ion mass shifts beginning at c3.

In both cases, glucuronyl modification results in a hydrophobic shift relative to the native peptide, with the N-terminal lysine modification being more abundant and eluting slightly earlier (more hydrophilic) than the modification on the lysine residue at the third position. Additional EThcD spectra of other glucuronyl-modified peptides are provided in the supplemental data.

In vitro forced glucuronidation of bsAb1

To assess the potential impact of glucuronidation, forced glucuronidation experiments were performed by incubating the bsAb1 for 24 hr at 37°C with varying concentrations of glucuronic acid sodium salt (0, 40, 100, and 140 mM). Intact mass analysis was used to measure the total levels of glucuronidation. As shown in the bottom spectrum of Figure 5, intact mass analysis does not resolve glycation and glucuronidation in the unstressed material. As a result, mono-glycation and mono-glucuronidation isoforms are observed as one peak with a mass shift of +166 Da, which lies between the mass shifts of glycation (+162 Da) and glucuronidation (+176 Da). However, in the stressed samples, the glucuronidated form of bsAb1 predominates over the glycated form, resulting in a more characteristic mass shift of glucuronidation (+176 Da). Therefore, the low levels of glycation in these samples had a minimum impact on the quantitation of glucuronidation. The total glucuronidation levels in the unstressed and stressed samples were 4.5%, 41.2%, 73.1%, and 83.8% as determined by intact mass analysis, respectively.

Figure 5.

Intact mass analysis of glucuronic acid-stressed material. From the bottom intact mass spectrum to top, bsAb1 incubated with 0, 40, 100, and 140 mM glucuronic acid, respectively.

The forced glucuronidated material underwent cIEF analysis to confirm the effects of its acidic shift. As indicated in Figure 6 and Table 2, the cIEF profiles resulting from glucuronidation stress indicate a significant reduction of the main peak’s intensity, decreasing from 55.2% to 7.7%. Conversely, glucuronidation led to the emergence of multiple acidic peaks with total values increasing from 39.8% to 92.3%, correlating with increased levels of glucuronic acid. Specifically, material incubated with 100 mM and 140 mM glucuronic acid exhibited three additional prominent acidic peaks. Furthermore, the total cIEF acidic region strongly correlated (r² = 0.9923) with the total glucuronidation measured by intact mass analysis as shown in Figure 7.

Figure 6.

cIEF electropherograms of bsAb1 after incubation with varying concentrations of glucuronic acid. The electropherograms, from bottom to top, show bsAb1 incubated with 0, 40, 100, and 140 mM of glucuronic acid, respectively. Acidic peaks 1–3 and basic peak 1, previously identified in unstressed bsAb1, are observed in all profiles. New acidic peaks (A–C) begin to emerge when bsAb1 was incubated with glucuronic acid concentrations of 40 mM and higher.

Table 2.

Integrated cIEF peak area % of bsAb1 incubated with 0, 40, 100, and 140 mM glucuronic acid.

Peak ID	0 mM	40 mM	100 mM	140 mM
New Acidic Peak C	ND	ND	2.5	4.5
New Acidic Peak B	ND	ND	6.1	9.7
New Acidic Peak A	ND	3.7	11.7	15.6
Acidic Peak 3	3.0	10.4	19.4	22.1
Acidic Peak 2	9.7	22.5	24.8	23.6
Acidic Peak 1	27.1	31.9	22.3	16.8
Main Peak	55.2	29.0	12.1	7.7
Basic Peak 1	5.0	2.5	1.1	ND
Sum of Acidic	39.8	68.5	86.8	92.3
ND = Not detected

Figure 7.

Total acidic cIEF peak area % versus the total glucuronic acid measured by intact mass analysis. Samples correspond to bsAb1 stressed with 0, 40, 100, and 140 mM glucuronic acid, respectively. Linear fit parameters (slope, intercept, and r²) are displayed on the plot.

Peptide mapping analysis of glucuronic acid-stressed bsAb1

Peptide mapping analysis was performed on all glucuronic acid-stressed bsAb1 after digestion using a combination of Glu-C and Asp-N enzymes to comprehensively characterize glucuronidation sites.

In Table 3, a numeric designation was assigned to each lysine residue based on its order in the amino acid sequence. The C-terminal lysine is present at levels <2% and therefore not included in this table. For peptides containing multiple-lysine residues, the designations are concatenated and separated by hyphens. For peptides containing one or more lysine residues, the chromatographic separation, identification, and subsequent quantitation of site-specific glucuronidation levels for each was challenging; therefore, for simplicity, the total peptide glucuronidation level was reported. As shown in Table 3, several lysine residues are resistant to glucuronidation (lysine residues 3*, 5, 9*, 10, 16, 35, and 47), while the remaining sites show a consistent increase with increased glucuronic acid stress.

Table 3.

Peptide mapping data of bsAb1 stressed with varying concentrations of glucuronic acid. The corresponding total glucuronidation by intact mass analysis for the control and each stress condition is presented for comparison. Lysine residues within the complementarity‑determining regions (CDRs) of bsAb1 are marked with an asterisk.

Glucuronic Acid Stress Concentration		Control (0 mM)	40 mM	100 mM	140 mM
Total Glucuronidation by Intact Mass Analysis		4.5%	41.2%	73.1%	83.8%
Lysine Designation	Subunit Location	% Glucuronidation by Peptide Mapping
1	Arm-A Fd’	NQ	NQ	NQ	0.1
2*	Arm-A Fd’	NQ	0.5	1.3	1.8
3*	Arm-A Fd’	NQ	NQ	NQ	NQ
4	Arm-A Fd’	NQ	0.4	1.0	1.3
5*	Arm-A Fd’	NQ	NQ	NQ	NQ
6–8	Arm-B Fd’	NQ	1.2	3.1	3.8
9*	Arm-B Fd’	NQ	NQ	NQ	NQ
10	Arm-B Fd’	NQ	NQ	NQ	NQ
11–13	Arm-A & Arm-B Fd’	0.3	2.1	4.5	4.8
14–15	Arm-A & Arm-B Fd’	0.1	1.0	2.0	3.0
16	Arm-A & Arm-B Fd’	NQ	NQ	NQ	NQ
17	Arm-A & Arm-B Fd’	NQ	0.6	1.5	2.1
18–19	Arm-A & Arm-B scFc	NQ	2.5	5.4	7.1
20	Arm-A & Arm-B scFc	NQ	0.3	0.7	1.1
21–22	Arm-A & Arm-B scFc	0.1	2.7	6.1	7.8
23	Arm-A & Arm-B scFc	NQ	0.2	0.4	0.6
24–26	Arm-A & Arm-B scFc	NQ	1.6	3.2	4.4
27–29	Arm-A & Arm-B scFc	NQ	0.1	0.5	0.8
30–31	Arm-A & Arm-B scFc	NQ	0.9	2.1	2.8
32	Arm-A & Arm-B scFc	NQ	0.4	0.9	1.2
33	Arm-A & Arm-B scFc	NQ	0.1	0.3	0.5
34	Arm-A & Arm-B scFc	NQ	0.1	0.3	0.3
35	Arm-A scFc	NQ	NQ	NQ	NQ
36–38	Arm-A LC	NQ	1.4	3.2	4.9
39	Arm-A LC	NQ	0.1	0.3	0.5
40–42	Arm-B LC	NQ	1.6	4.0	5.4
43	Arm-A & Arm-B LC	NQ	0.2	0.6	1.0
44	Arm-A & Arm-B LC	NQ	0.5	1.4	1.9
45–46	Arm-A & Arm-B LC	NQ	1.0	2.7	3.6
47	Arm-A & Arm-B LC	ND	ND	ND	ND
48	Arm-A & Arm-B LC	0.2	1.1	2.3	3.2
49–50	Arm-A & Arm-B LC	0.1	2.5	5.5	7.6
51	Arm-A & Arm-B LC	NQ	0.5	1.1	1.5

NQ = Not quantitated due to low signal intensity.

In evaluating the sequence features associated with glucuronidation, no specific amino acid motif, including the KG motif, was found to correlate with increased susceptibility to glucuronidation. The bsAb1 molecule contains four KG motifs, yet their glucuronidation levels under 140 mM glucuronic acid stress range from 0% to approximately 5%, demonstrating that the presence of a KG sequence does not inherently predispose a lysine residue to glucuronidation. Instead, the variability in glucuronidation across lysine residues appears to be influenced by other factors, such as lysine solvent exposure and structural accessibility to glucuronic acid due to neighboring negatively charged residues. Also, peptides with higher glucuronidation levels typically contain multiple modified lysine residues. Of the four complementarity‑determining region (CDR) lysine residues in bsAb1 (indicated by an asterisk in Table 3), only the lysine residue in Arm-A (designated as 2*) showed an increase in glucuronidation under stress. Even at 140 mM glucuronic acid stress, which increased total glucuronidation to 84%, modification at this site remained below 2%.

Subunit mass analysis of glucuronic acid-stressed bsAb1

Subunit mass analysis was performed on all glucuronic acid-stressed samples. As shown in the top panel of Figure 8, samples were treated with PNGase F to remove N-linked glycans, CPB to remove C-terminal lysine, IdeS^24,42,43 to cleave below the hinge region, and dithiothreitol (DTT) to reduce inter-chain disulfide bonds, resulting six distinct subunits for bsAb1: Arm-A Fd,’ Arm-B Fd,’ Arm-A light chain (LC), Arm-B LC, Arm-A single chain Fc (scFc), and Arm-B scFc. As shown in the bottom panel of Figure 8, glucuronidation levels of all these subunits increased linearly with the increase of glucuronic acid stress; therefore, the two scFc subunits can serve as surrogate markers for monitoring glucuronidation levels across the entire bsAb1. Results of the scFc glucuronidation analysis are discussed below.

Figure 8.

Subunit mass analysis of glucuronidated bsAb1. The top panel illustrates the sample preparation process and the resulting subunits (Arm-A and Arm-B LC, Fd′, and scFc) following PNGase F, CPB, IdeS, and DTT treatment. The bottom panel illustrates quantitative subunit mass analysis results for total glucuronidation levels for each subunit under increasing glucuronic acid stress (0, 40, 100, and 140 mM). Linear correlations among subunit regions were observed, demonstrating consistent modification trends across both antibody arms.

Bioactivity of glucuronic acid-stressed bsAb1

Biological characterization, including antibody-dependent cell-mediated cytotoxicity (ADCC), antigen binding, FcγRIIIa, and FcRn binding assays, was performed to evaluate the structure/function relationships pertaining to glucuronic acid stress. Given that both Arm-A and Arm-B of the bsAb1 contain one or more lysine residues within their CDR regions, along with additional lysine residues in the Fc region that could be susceptible to glucuronidation, the structural integrity and functionality of each arm were thoroughly examined. The lysine sites that are in proximity to the antigen and Fc binding sites are presented in Figure 9.

Figure 9.

Molecular modelling of antigen and Fc binding sites in proximity to lysine residues. Lysine residues within the complementarity‑determining regions (CDRs) of bsAb1 are marked with an asterisk.

The impact of increasing glucuronidation on ADCC activity is illustrated in Panel A of Figure 10 and Table 4. Notably, glucuronidated bsAb1 displayed no meaningful change in ADCC activity, ranging from 107% to 99%, which falls within the expected assay variability, indicating that increased glucuronidation levels have a negligible effect on the ADCC activity. Similarly, the antigen-binding activity of Arm-B remained stable, even as total glucuronidation levels increased from 4.5% to 83.8% (Figure 10, Panel B and Table 4). Additionally, FcγRIIIa binding and FcRn binding remained stable even as total glucuronidation reached nearly 100% (Table 4).

Figure 10.

Correlation of ADCC (A) and antigen binding activity (B) with total glucuronidation. Linear fit parameters (slope, intercept, and r2) are displayed on each plot.

Table 4.

Table of glucuronic acid stress samples with respective binding assays.

Bioactivity	Attribute	Total Glucuronidation by Intact Mass Analysis (%)
		4.5	41.2	73.1	83.8	98.9
Arm-A ADCC	Arm-A Fab & Fc Functionality	107	105	102	99	NT
Arm-B Binding	Arm-B Fab Functionality	101	111	99	102	NT
FcγRIIIa	Fc Functionality	96	NT	NT	NT	107
FcRn	Fc Functionality	104	NT	NT	NT	105
NT = Not tested

Comprehensive analyses, including SEC, CE-SDS (R and NR), CD spectroscopy, SV-AUC, and DSC were also performed on select glucuronic acid-stressed material (supplemental data). These results confirmed that glucuronidation had no impact on bsAb1 aggregation, fragmentation, or higher order structure, despite nearly 20-fold increase in total glucuronidation.

Glucuronidation and glycation measured by subunit mass analysis

For simplicity, this analysis focuses on the scFc regions to quantify glycation and glucuronidation; Figure 11 shows the subunit mass spectrum for the scFc regions of bsAb1. At the subunit level, the bsAb1 can be clearly differentiated into Arm-A and Arm-B, along with their mono-glycated and mono-glucuronidated isoforms based on their molecular masses. As shown in Figure 11, glycation levels were quantified at 0.3% and 0.4%, while glucuronidation levels were quantified at 0.2% and 0.6% for scFc Arm-A and Arm-B, respectively. Since glycation and glucuronidation occur randomly across surface lysine residues, subunit mass analysis of the scFc regions offers valuable insights into the glycation and glucuronidation levels of bsAb1.

Figure 11.

Subunit mass spectrum of the scFc fragments of bsAb1. The mass range 20,170– 20,280 Da is zoomed in 10-fold to highlight the baseline-resolved peaks representing the glycated (blue highlight) and glucuronidated (red highlight) species.

Subunit mass analysis of glycation and glucuronidation only takes approximately 15 minutes per sample, thus providing a higher throughput alternative to peptide mapping analysis for characterizing in-process samples, drug substance and drug products.

Qualification results for total percent scFc glucuronidation by subunit mass analysis

Following ICH-Q2(R1), the performance of this subunit method was further qualified by evaluating method specificity, precision (including repeatability and intermediate precision), as well as the limits of quantitation (LOQ) and detection (LOD). For simplicity, only the data from the scFc region of each arm of bsAb1 are discussed.

Specificity was evaluated by assessing glucuronidation of bsAb1 in comparison to its formulation buffer to identify potential matrix interference. In this context, specificity is defined as the method’s ability to unambiguously quantify the analyte amidst expected components. As described in Table 5, no peaks exceeding the LOQ of 0.6% were detected in blank that contained the formulation buffer matrix, indicating no interference with the glucuronidation measurements for the bsAb1 analyte.

Table 5.

Summary of qualification results for scFc glucuronidation measurements from subunit analysis. The data presented regarding specificity, LOD, and LOQ are derived specifically from the analysis of bsAb1. The repeatability and intermediate precision data include bsAb1 and its respective parental molecules.

Parameter	Test Result
Specificity	No peaks above 0.6% were detected that could interfere with measurement of total percent scFc glucuronidation.
LOD*	0.2
LOQ**	0.6
Repeatability (n = 6)	RSD: 14%
Intermediate Precision (n = 24)	RSD: 20%

RSD = Relative standard deviation.

*The percent total scFc glucuronidation at the LOD has a signal to noise ratio of 3.

**The percent total scFc glucuronidation at the LOQ has a signal to noise ratio of 10.

Intermediate precision was evaluated by examining intra-laboratory variations, including different analysts, days, and instrumentation. This assessment utilized combined precision data from two analysts over two days (n = 24) for bsAb1 and its parental molecules. The intermediate precision data for the percentage total scFc glucuronidation are presented in Table 2, showing that the intermediate precision for the total scFc glycation was ≤20% for all species examined.

Repeatability, which reflects precision under the same operating conditions over a short time frame, was assessed by testing six sample replicates to determine the highest variability. The worst-case scenario was chosen to represent repeatability in our analysis. As shown in Table 5, the relative standard deviation (RSD) for the repeatability of the percentage of total scFc glucuronidation was ≤14%.

The LOQ and LOD for the percentage of total scFc glucuronidation in bsAb1 and its two parental molecules were determined based on the signal-to-noise (S/N) ratio of the scFc glucuronidation peaks in the deconvoluted mass spectra. LOQ and LOD were defined at S/N ratios of 10 and 3, respectively. As shown in Table 5, the LOQ was 0.6% with an S/N of 10, while the LOD for the percentage of total scFc glucuronidation was 0.2% with an S/N of 3.

Discussion

Glucuronidation has been identified as a new acidic charge variant in a bsAb. Definitive evidence for site-specific glucuronidation in bsAb1 was obtained through peptide mapping using accurate mass spectrometry coupled with EThcD fragmentation. This analysis was performed on enriched CEX-fractionated material, which significantly facilitated the detection of the glucuronidation. Identifying glucuronidation in unenriched material, particularly at low levels, would have presented considerable challenges.

The in vitro enhancement of forced glucuronidated material successfully replicated the effects observed in the acidic region of the cIEF profile. This modified material was subsequently used to investigate the structure/function relationship and its impact on various binding activities. Antigen binding, ADCC activity, FcγRIIIa binding and FcRn binding remained consistent despite nearly 20-fold increase in total glucuronidation, indicating that glucuronidation did not adversely affect the biological activities of bsAb1. Although the impact of glucuronidation may vary among antibody products, the analytical strategy described in this study is broadly applicable.

Both glucuronidation and glycation contribute to increased heterogeneity in the acidic region of the charge variant profile, which can complicate the characterization of therapeutic proteins. Intact mass analysis, a widely used analytical technique in the pharmaceutical industry for quantifying total glycation,¹¹ has inherent limitations in distinguishing between glucuronic acid and glucose modifications when both are present at similar levels. The mass difference between glucuronidation and glycation (~14 Da) is minimal relative to the total mass of a typical monoclonal antibody (~150 kDa), increasing the likelihood of misidentifying one modification as the other. The quadrupole time-of-flight mass spectrometer used in this study for the intact mass analysis did not provide sufficient resolution to confidently differentiate between these two modifications in unstressed material. However, intact mass analysis accurately quantified the forced glucuronidated material because glucuronidation levels were substantially higher than glycation, and the intact mass signal from the glucuronic acid-modified form significantly exceeds that of the glucose-modified form. In typical scenarios, where both modifications are present at similar levels, the resulting peak reflects a weighted average of the two masses, complicating accurate identification and quantitation.

The subunit mass method provides higher specificity than the intact mass method and greater throughput compared to peptide mapping. Although peptide mapping can quantify glucuronidation accurately, it is time consuming and less practical for routine analysis. In addition, subunit mass can differentiate glucuronic acid from glycation and has the benefit of discerning homologous subunits of bispecific antibodies. As a result, the subunit mass method was qualified to enable efficient routine monitoring of glucuronidation and glycation in antibody products to ensure process consistency.

After method qualification, the subunit mass method is now routinely implemented as part of our antibody process-monitoring strategy. During process characterization and comparability assessments, subunit method enables rapid screening of glucuronidation and glycation across development lots, providing assurance of process consistency without the need for more time-consuming peptide mapping. It is worth noting that the subunit method can also differentiate glucuronidation between the 2 arms for bsAb, which is not possible by peptide mapping for peptides that share the same amino acid sequences.⁴⁴

Notably, using the subunit method, we observed that glucuronidation is commonly present in antibody products. This may have been previously overlooked due to the limited resolution of intact mass analysis, which cannot fully distinguish glucuronidation from glycation. Although the impact of glucuronidation may vary among antibody products and this study focused on a single bsAb, our analytical strategy is broadly applicable to other molecules.

Finally, we evaluated potential safety and immunogenicity risks. To date, there are no published clinical findings linking lysine glucuronidation on antibodies to adverse safety or immunogenicity outcomes. In unstressed antibodies, glucuronidation typically occurs at <0.3% per site, well below thresholds expected to affect safety or pharmacokinetics. Based on the totality of evidence, glucuronidation is classified as a non-critical quality attribute for bsAb1.

Materials and methods

Reagents

D-(+)-Glucuronic acid sodium salt monohydrate was purchased through Sigma-Aldrich. Sodium bicarbonate was purchased through BDH Chemicals. IdeS (FabRICATOR®) and EndoS (IgGZERO®) were purchased from Genovis. Glu-C endoproteinase was purchased from Thermo Scientific. Reagents used for cIEF analysis were purchased from ProteinSimple™/Bio-Techne® unless stated otherwise. All other reagents were purchased from Thermo Fisher Scientific or Sigma-Aldrich unless stated otherwise. All reagents were analytical reagent grade or mass spectrometry grade and were used without further purification. All solutions were prepared with Milli-Q water. All IgG1 molecules (bsAb1, parental Arm-A, and parental Arm-B) were manufactured by Johnson & Johnson Innovative Medicine.

cIEF

Samples were pre-treated with CPB (1:20 w/w CPB:protein ratio; Sigma-Aldrich, catalog number C9584) and incubated at room temperature for 30 minutes to remove C-terminal lysine. Analysis was conducted on a ProteinSimple™ iCE3™ analyzer using a 100-µm inner wall-coated silica capillary with an outer wall polyimide coating. An anolyte solution of dilute phosphoric acid and methylcellulose, along with a catholyte solution of sodium hydroxide and methylcellulose, was employed, and a defined mixture of 3–10 and 8–10.5 Ampholytes (GE HealthCare) were used. Protein was detected at UV 280 nm and pI markers of 7.40 and 9.46 were used for calibration.

Fractionation of bsAb1

Fractionation of bsAb1 was performed using an Agilent 1260 HPLC system with a strong CEX column (ProPac™ WCX-10, 10 µm, 4 × 250 mm, Thermo Scientific™). Fractions were eluted over 30 minutes at 35°C using a linear pH gradient with CX-1 pH 5.6 Gradient Buffer A and CX-1 pH 10.2 Gradient Buffer B (Thermo Scientific™). After multiple CEX runs, equivalent fractions were pooled and exchanged into formulation buffer prior to characterization.

Peptide mapping analysis

Each sample (250 μg) was desalted and buffer exchanging into a denaturing buffer (6 M guanidine, 50 mM Tris, pH 8.0, 5 mM EDTA) using a 10 KDa molecular weight cutoff (MWCO) Amicon filter, yielding a final concentration of approximately 2.5 mg/mL. Samples were reduced with 25 mM DTT at 37°C for 15 minutes, then cooled to room temperature. The reduced samples were alkylated with 60 mM sodium iodoacetate at 25°C in the dark for 15 minutes, and the alkylation reaction was terminated by adding excess DTT. The samples were buffer exchanged into digestion buffer (50 mM Tris, pH 8.0) using a PD MiniTrap™ G10 column (Cytiva). Glu-C was added at a 1:25 (enzyme:protein) ratio and incubated at 37°C for 3 hours. Asp-N was subsequently added at a 1:100 (enzyme:protein) ratio and incubated at 37°C for an additional 2 hours. The digested samples were cooled to room temperature, and the digestion was terminated with 3.0 μL trifluoroacetic acid. The digested samples were then analyzed by LC-MS/MS with EThcD and HCD fragmentation using an Agilent 1290 HPLC coupled to an Orbitrap Eclipse™ Tribrid™ mass spectrometer (Thermo Scientific™). Peptide mapping data were analyzed using Protein Metrics software.

Glucuronic acid-stressed bsAb1

The dilution of bsAb1 to 52 mg/mL was performed using 125 mM sodium bicarbonate (pH 8.3) with glucuronic acid at starting concentrations of 0, 40, 100 and 140 mM. After incubation at 37°C for 24 hours, samples were buffer exchanged into formulation buffer through a MWCO filter to remove excess, unbound glucuronic acid.

Intact mass analysis

Samples were deglycosylated by incubation with PNGase F (QA-Bio, catalog number E-PNG01) for overnight at 37°C. The treated samples were analyzed by LC-MS on a Waters Premier Acquity with a reversed phase C4 column coupled to a Xevo G3 mass spectrometer. Data were collected and analyzed by MassLynx software. The raw spectra were processed through MaxEnt 1 deconvolution and centered to get the intensities of each peak. Total levels of glucuronidation were determined by summing the relative abundance of each form (e.g., mono-glucuronidation, di-glucuronidation, tri-glucuronidation) relative to the sum total peak height of both unmodified and modified forms, and expressed as a percentage.

Subunit mass analysis

Sample were diluted with 100 mM Tris buffer, pH 7.5 to a final concentration of 1 mg/mL and incubated with PNGase F for overnight at 37°C. IdeS enzyme (1 unit/ug of protein) and CPB were added and the samples were incubated at 37°C for 1 hour. The digested samples were reduced with 50 mM DTT at 37°C for 15 min and the reaction was quenched with 10% (v/v) of 1 M HCl. Treated samples were analyzed on a Waters H-class Acquity with a reversed phase C4 column coupled to a Xevo G2-XS mass spectrometer. Data were collected and analyzed by MassLynx software. Raw spectra were processed through MaxEnt 1 deconvolution and centered to get the intensities of each peak.

Bioactivity assays

Antigen binding activities were assessed using competitive time-resolved fluorescence resonance energy transfer (TR-FRET) assays. In these assays, varying concentrations of unlabeled bsAb1 compete with donor fluorophore-labeled bsAb1 for binding to an acceptor fluorophore-labeled antigen. When the donor fluorophore is excited, energy is transferred to the bound acceptor fluorophore, resulting in fluorescence resonance energy transfer (FRET), which is detected using a microplate reader designed for time-resolved fluorescence measurements. Fluorescence signals were plotted against sample concentrations and analyzed using a 4-parameter curve fit. The concentration required to achieve 50% of the maximum TR-FRET signal (EC50) was calculated for each sample. The relative binding activity of each sample was then determined by the ratio of the EC50 of the reference sample to that of the tested samples, with potency expressed as a percentage relative to the reference material.

The ADCC bioactivity of bsAb1 was assessed using a cell-based reporter assay, which uses antigen-expressing target cells and engineered effector cells that express the FcγRIIIa receptor. BsAb1 binds to antigens on the cell-surface of target cells. The FcγRIIIa receptors on the effector cells recognize the target cell-bound antibodies, which triggers a signal transduction cascade and activates firefly luciferase expression. Cells were lysed and the luciferase activity in the effector cells was quantified using a microplate reader designed for luminescent measurements. The signal obtained was plotted against bsAb1 concentration and analyzed by a 4-parameter curve fit. The potency of test articles was calculated relative to the bsAb1 reference material and expressed as a percentage.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Abbreviations

ADCC

Antibody-dependent cell-mediated cytotoxicity

CD

Circular dichroism

CDR

Complementary-determining region

CE-SDS

Capillary electrophoresis-sodium dodecyl sulfate

CEX

Cation exchange

CID

Collision-induced dissociation

cIEF

Capillary isoelectric focusing

CPB

Carboxypeptidase B

DSC

Differential scanning calorimetry

EThcD

Electron-transfer/higher-energy collision dissociation

Fc

Fragment crystallizable

Fd’

Defined as the N-terminal fragment of the HC after cleavage below the hinge region by IdeS protease

HC

Heavy chain

HCD

Higher-energy collisional dissociation

HMWs

High molecular weight species

IEX

Ion exchange

LC

Light chain

LC-MS

Liquid Chromatography-Mass Spectrometry

LMWs

Low molecular weight species

mAb

Monoclonal antibody

pI

Isoelectric point

PK

Pharmacokinetics

PTM

Post-translational modification

scFc

Single chain Fc

SEC

Size exclusion chromatography

SV-AUC

Sedimentation velocity analytical ultracentrifugation

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2025.2612471