Abstract
This technical note demonstrates intact- and subunit-based imaged capillary isoelectric focusing (icIEF)-UV/MS workflows on the Intabio ZT system for comprehensive characterization of monoclonal antibodies (mAbs) under thermal and oxidative stress conditions. The intact icIEF-UV/MS analysis comprehensively characterizes charge variants and identifies proteoforms, while subunit icIEF-UV/MS analysis reveals the charge heterogeneity of F(ab’)2 and Fc complexes and localizes the post-translational modifications (PTMs) observed on the intact level to light chain (LC), Fd’, or Fc/2 subunit.
The single platform, integrated icIEF-UV/MS workflow enables sensitive detection, rapid charge separation, and confident identification of biotherapeutics and their proteoforms. The combined capabilities of intact and subunit icIEF-UV/MS workflows integrate isoelectric point (pI) with intact/subunit mass information, enabling PTMs identification and localization to distinct mAb domains and enhancing characterization capabilities essential for biotherapeutics development.
Key features of the intact and subunit icIEF-UV/MS workflows
- Multi-level icIEF-UV/MS analysis: The Intabio ZT system enables integrated icIEF-UV/MS analysis at intact and subunit levels, allowing global PTMs assessment, accurate PTMs localization to the subunits, and increased confidence in proteoform identification
- Single-pot sample preparation for subunit icIEF-UV/MS analysis: A single-pot digestion and reduction reaction allows subunit icIEF-UV/MS analysis, for pI profiling of F(ab’)₂ and Fc and PTMs localization to LC, Fd’, or Fc/2 subunit
- Streamlined icIEF-UV/MS workflows with advanced software tools: Biologics Explorer software offers powerful data visualization and a time-resolved deconvolution function for identifying proteoforms with subtle pI and mass differences
Introduction
Antibody-based biotherapeutics are highly heterogeneous due to various enzymatic and chemical modifications. In-depth characterization of PTMs is essential for critical quality attributes (CQAs) assessment.1 Forced degradation studies are routinely performed during biotherapeutics development to evaluate stability, identify potential degradation pathways, and support formulation and analytical development.2 Since different mAb domains are responsible for antigen binding or immune activation, accurate PTMs localization is crucial for maintaining product consistency and ensuring safety, identity, strength, purity, and quality (SISPQ).3
Previously, we have demonstrated the capabilities of the Intabio ZT system for comprehensive characterization of charge variants and PTMs identification on the intact level.4,5 Compared to traditional fractionation-based workflows for assessing charge heterogeneity, the integrated icIEF-UV/MS platform offers a faster, more precise, and more confident way for charge variants analysis and proteoforms identification. This is achieved with simple sample preparation and only 30 minutes of data acquisition time, for a reduction in labor and time to streamline and accelerate biotherapeutic development.6
As an alternative to peptide mapping, subunit-based mAb analysis can be an efficient approach, that provides rich sequence information with a simplified workflow. In a previous study, cIEF of digested mAbs enabled high-resolution profiling of charge variants distributions of subunit complexes.7 This technical note demonstrates the applications of integrated icIEF-UV/MS analysis on both intact and subunit levels to analyze unstressed and stressed trastuzumab samples, enabling comprehensive, high-confidence monitoring of charge variants and proteoforms.
Methods
Forced degradation of trastuzumab: Heat-stressed trastuzumab was generated by incubating a 20 mg/mL trastuzumab solution at 40oC for 1 month. For oxidative stress, 5 mg/mL of trastuzumab was incubated in 20 mM sodium acetate buffer (pH 5.3) with 1 mM 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH, Sigma-Aldrich) at 37°C for 22 hours. Following incubation, all samples were immediately desalted using Zeba spin desalting columns (Thermo Scientific) and stored at - 80oC until analysis.
Platform sample preparation for intact icIEF-UV/MS analysis: Intact desalted samples were mixed with the master mix solution to achieve final protein concentrations of 0.4-0.8 mg/mL, containing 15 mM arginine (Sigma-Aldrich), 3% of each Pharmalyte 5-8 and 8-10.5 (Cytiva), and 6.3 μg/mL of each pI marker with estimated pI values of 5.52 and 9.68.
Single-pot digestion and reduction for subunit icIEF-UV/MS analysis: 50 μg of samples were incubated with 50 units of FabRICATOR enzyme (Genovis), 20 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl, Thermo Scientific), and 50 mM arginine at 37 °C for 1 hour. Digested and reduced samples were then mixed with the master mix solution to achieve a final protein concentration of 0.25 mg/mL, containing 15 mM arginine, 6% Pharmalyte 3-10 (Cytiva), and 6.3 μg/mL of each pI marker with estimated pI values of 4.05 and 9.99.
icIEF-UV/MS analysis: The icIEF-UV/MS analysis was performed using the Intabio ZT system (SCIEX). UV absorbance was recorded at 1 Hz during analysis. Focusing was performed in 3 steps over a total duration of 6.5 minutes: 60 seconds at 1,500 V, 60 seconds at 3,000 V, and 270 seconds at 4,500 V. Samples were then introduced into the ZenoTOF 7600 system at a 3 μL/min flow rate with a tip voltage of 5,500 V and a delta voltage of 3,000 V for 10 minutes. The MS data were acquired with ZenoTOF MS method, and the key parameters are shown in Table 1.
Intact icIEF-UV/MS analysis of heat-stressed trastuzumab
Trastuzumab was incubated at 40oC for 1 month to generate the heat-stressed material. Intact icIEF-UV/MS analysis was performed on both unstressed and heat-stressed samples to assess and identify changes in charge heterogeneity (Figure 2). As shown in the icIEF-UV profiles, the unstressed sample contains 2 major acidic variants with a total abundance of 38.3%, as calculated from the UV peak area (Figure 2A). The icIEF-UV profile of the heat-stressed sample shows 5 acidic variants with a total abundance of 67.4% (Figure 2D), a noticeable increase in the number and abundance of acidic variants compared to the unstressed sample. This increase in acidic species is due to an elevated level of thermally enhanced modifications, such as deamidation. Moreover, the abundance of Basic 1 peak relative to the main peak was increased in the stressed sample.
The corresponding icIEF-MS profiles confirmed that each charge variant peak observed in the icIEF-UV profile had a matching peak, demonstrating the high-resolution separation maintained during the chemical mobilization process (Figures 2B and 2E). Due to the mobilization process, the peaks appeared in the reverse order in the icIEF-MS profiles (Figures 2B and 2E) compared to the corresponding icIEF-UV profiles (Figures 2A and 2D), with basic variants being detected earlier and acidic variants later. The Biologics Explorer software provides excellent data visualization with time-resolved deconvolution function, enabling quick visualization and comparison between the unstressed and heat-stressed samples.
In the unstressed sample, the most abundant proteoform was identified as the G0F/G1F glycoform without the C-terminal lysine, with a deconvoluted mass of 148,219 Da (Figure 2C). High mass accuracy (<20 ppm) was achieved with internal mass calibration using the clusters of arginine, which was also an icIEF cathodic spacer in the master mix solution. Two basic variants, B2 and B1, were attributed to the species carrying a C-terminal lysine (Lys) and N-terminal cyclization of glutamic acid (PyroQ), respectively. The acidic modifications included glycation (+Hex), terminal sialylation (+NeuAc), and deamidation. The deconvoluted results of the heat-stressed sample revealed up to 5 deamidation events (estimated based on pI shift), contributing to the increased abundance of the acidic variants (Figure 2F). The increase in the relative abundance of the Basic 1 peak in the stressed sample was attributed to promoted N-terminal cyclization reaction (PyroQ).
Single-pot digestion and reduction for subunit icIEF-UV/MS analysis
The icIEF-UV/MS analysis of biotherapeutics on the subunit level enables the localization of PTMs observed on the intact level to specific subunits. In this work, a single-pot digestion and reduction method was developed for subunit icIEF-UV/MS analysis of mAbs (Figure 4). The mAb sample was incubated with the FabRICATOR enzyme (IdeS), which cleaves the sequence below the hinge region of the HC, along with the reducing agent TCEP to reduce the interchain disulfide bonds, leading to the formation of non-covalently bound F(ab’)2 and Fc complexes. During subunit icIEF-UV/MS analysis, TCEP was present to maintain the reduced state of the interchain disulfide bonds without the need for alkylation. In the focusing step, the F(ab’)2 and Fc complexes were separated based on their pIs. During mobilization, these complexes remained intact and were electrochemically mobilized until reaching the mobilizer junction toward the electrospray ionization (ESI) tip. The non-covalently bound complexes were denatured during ESI, leading to the detection of LC, Fd’, and Fc/2 subunits by MS. This single-pot approach allows simple sample preparation for streamlined subunit icIEF-UV/MS analysis, providing pI information of the F(ab’)2 and Fc complexes and accurate mass measurement of each subunit, enhancing the proteoforms identification and PTMs localization.
Subunit icIEF-UV/MS analysis of heat-stressed trastuzumab
Subunit icIEF-UV/MS analysis was performed on both unstressed and heat-stressed samples to localize the PTMs observed on the intact level to each subunit (Figure 5). The icIEF-UV profiles demonstrate high-resolution separation of charge variants with 2 distinct groups of peaks corresponding to the F(ab’)2 and Fc complexes (Figures 5A and 5D). In the unstressed sample, F(ab’)2 contained 1 basic variant (Fab-B) and 2 acidic variants (Fab-A1 and Fab-A2) in addition to the main species, while Fc had 1 basic and 1 acidic variants (Fc-B and Fc-A1 in Figure 5A). Similar to the observations from the intact icIEF-UV/MS analysis, the heat-stressed sample showed an increased level of the acidic variants in the subunit analysis (Figure 5D). The increase in the basic variant observed on the intact level was localized to the F(ab’)2 complex (Fab-B in Figure 5D).
During the ESI process, the F(ab’)2 and Fc complexes were dissociated into the LC, Fd’, and Fc/2 subunits under denaturing conditions. In the unstressed sample, the glycation (+Hex) events were localized to the LC and Fd’ subunits, while deamidation was detected for all 3 subunits (Figure 5C). The basic variant corresponds to N-terminal cyclization (+PyroQ) of the Fd’ subunit. For the Fc/2 subunit, 6 glycoforms were detected under the main peak, including the low-abundant G0, G0F-GlcNAc, M5, and an aglycosylated species. In the acidic region, terminal sialylation (+NeuAc) was observed, along with deamidation. In the heat-stressed sample, the abundance of the deamidated species was increased for all 3 subunits (Figure 5F). The increased abundant of PyroQ was observed and localized to the Fd’ subunit. These results demonstrate that the subunit icIEF-UV/MS workflow provides high-resolution charge separation of F(ab’)2 and Fc, confident subunit identification, and accurate PTMs localization.
Intact and subunit icIEF-UV/MS analysis of AAPH-stressed trastuzumab
Trastuzumab was incubated with AAPH to generate oxidative-stressed samples. Intact and subunit icIEF-UV/MS workflows were employed to investigate the associated heterogeneity induced by oxidation (Figure 6).
The intact icIEF-UV electropherograms of the unstressed and AAPH-stressed trastuzumab revealed an increased level of the acidic variants and heterogeneity within the major charge variant peaks upon oxidative stress (Figure 6A). This is attributed to the minor pI shifts induced by oxidation. Each oxidation event contributes approximately +16 Da on the intact (~150 kDa) protein, which is not easily discernible at the intact level due to the relatively minor mass change. As a result, the detection of oxidation events is challenging due to the minimal changes in both pI and mass. However, the high-resolution separation and time-resolved deconvolution capabilities of the icIEF-UV/MS platform enabled the detection of these subtle modifications.
The minor mass shift within the main peak was observed in the intact deconvoluted mass map of the AAPH-stressed sample (Figure 6B). Scan-by-scan deconvoluted spectra revealed a clear mass increase across the main peak from the basic front to the acidic end (Figure 6C).
Subunit analysis provided further insights into the localization of oxidation sites and offered improved mass resolution, since a +16 Da shift is more discernible on a subunit than on the intact molecule. The subunit icIEF-UV profile of the AAPH-stressed sample showed a more pronounced split peak in the Fc complex (circled in Figure 6D), suggesting that most of the oxidative changes occurred within the Fc domain. This was further confirmed by the MS data, which revealed the presence of up to 2 oxidation events in the Fc/2 subunit (Figures 6E and 6F). The combination of intact and subunit icIEF-UV/MS analysis enabled the identification of proteoforms with subtle pI and mass shifts, providing detailed insights into the oxidative modifications of trastuzumab under stress conditions.
Conclusion
- The Intabio ZT system offers 30-minute, integrated icIEF-UV/MS workflow on both intact and subunit level, for high-resolution charge separation, sensitive proteoform detection, and confident PTM identification and localization, providing a faster, easier, and more direct alternative to conventional fractionation-based workflows
- A simple, single-pot sample preparation method was developed for subunit icIEF-UV/MS analysis, eliminating the need for alkylation while preserving the reducing state of the disulfide linkages
- Non-covalently bound F(ab’)2 and Fc complexes were maintained during icIEF, providing pI information, while these complexes were dissociated into the LC, Fd’, and Fc/2 subunits under denaturing ESI conditions, leading to confident localization of PTMs observed on the intact level to specific subunits
- Intact icIEF-UV/MS analysis of the heat-stressed sample revealed that thermal degradation promoted N-terminal cyclization within the Fd’ subunit and deamidation across all 3 subunits
- Subunit icIEF-UV/MS analysis of the AAPH-stressed sample showed that oxidation predominantly occurred on the Fc/2 subunit
- SCIEX OS software allows internal mass calibration using arginine clusters to achieve consistently high mass accuracy, while Biologics Explorer software offers excellent data visualization tools and powerful time-resolved deconvolution to characterize proteoforms with subtle pI and mass differences
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