Enhancing Biopharmaceutical Efficiency: Intact-Level PTM Identification and Charge Variant Analysis

Intact-level analysis maintains the original molecular form versus analytical strategies like peptide mapping that break apart the molecule and so require many more sample preparation steps. This additional sample preparation adds a significant amount of time to the development timeline but can also introduce undesired protein modifications or artifacts.  Intact-level analysis requires minimal sample preparation and can minimize any potential PTMs induced during the sample preparation process and/or fragmentation. In addition, being able to perform characterization assays at an intact level provides a quick insight into molecular heterogeneity and allows for confident PTM identification much earlier in the development pipeline where this information can be used to guide the overall analytical strategy for top clones.  

With traditional liquid chromatography-mass spectrometry (LC-MS) intact analysis, it is hard to resolve isomers, since some combinations of different PTMs can result in similar or identical delta masses. For example, glycation events can’t be identified without additional sample treatment. Additionally, some proteoforms with similar mass are challenging to identify without further separation based on another physical attribute. For example, deamidation at the intact level is almost impossible to confidently identified using traditional LC methods. It is also hard to confidently detect and identify low abundance proteoforms, since those lower signals detection can be interfered with background noise. Analyzing complex molecules using only mass spectrometry data poses challenges, as lacking additional dimensional information makes it difficult to narrow down potential candidates for each mass identification. icIEF-UV/MS technology offers information in two dimensions: charge separation and mass differentiation. Various proteoforms in heterogeneous biotherapeutic molecules can be separated into charge groups, which simplifies the data analysis process before mass differentiation.

Charge variant analysis is used to detect and analyze the charge heterogeneity present in biotherapeutic molecules that are often caused by PTMs during production and storage. For example, glycation and deamidation will cause an acidic shift of the isoelectric point (pI) values, while unprocessed C-terminal lysine or succinimide intermediate will cause a basic shift. It is a critical quality attribute to monitor since the charge variants could significantly impact the stability, efficacy and safety of the biotherapeutics.

icIEF is one of the most common technologies for high-resolution charge variant analysis. With the pH gradient established by the ampholytes, proteoforms with different charges get focused into groups according to their pI. icIEF applies to multiple molecule modalities, including antibodies, antibody-drug conjugates or fusion proteins, and can be used for different purposes, such as stability monitoring and quality assessment. The data generated with icIEF technologies is used to estimate the pI values and quantify the percent compositions of each charge group. 

The Intabio ZT system consists of an icIEF-UV separation system, which directly couples to the ZenoTOF 7600 system for intact MS analysis. The system uses microfluidic chip-based integrated icIEF-UV/MS technology.

The protein of interest is introduced with ampholytes and pI markers into the sample channel and separated by icIEF with the whole channel monitored under UV 280 nm. After separation, an electrolyte is introduced to recharge focused charge variants and electrochemically mobilize them, which better maintains the separation resolution established during focusing, compared to pressure-driven mobilization. With on-chip ESI, each charge variant is directly sprayed into and detected by MS for intact-level analysis.

This technology allows for the significant simplification of workflows involved in charge variant identification. With minimal method optimization, sample preparation and no scale-up or protein purification needed, this analysis can shorten the workflow from weeks to hours. The existing icIEF separation conditions can be adopted for this icIEF-UV/MS analysis with MS-compatible alternatives. The sample only needs to be desalted before analysis. Each injection uses ~20 ug of protein and takes 30 minutes to be analyzed. Compared to LC-MS, low-abundance proteoforms can be identified confidently – since these proteoforms get locally concentrated during the focusing process and can be detected by MS on the intact level.

The data generated includes icIEF-UV profiles and a vast amount of MS data. The icIEF-UV profiles generated on the Intabio ZT system are comparable to other existing icIEF platforms. In addition, the MS data is generated to identify and annotate each charge variant. Due to the mobilization process, basic variants are mobilized and detected by MS first, followed by the acidic variants. The data contains information on three dimensions: pI, mass and intensity.

The modifications are identified according to the delta mass and delta pI value compared to the most intense proteoform in the sample. This two-dimensional analysis allows for the annotation of isomers that cannot be identified by traditional LC-MS intact analysis.

For example, for a monoclonal antibody molecule, compared to the mass of G0F/G1F glycoform, G0F/G1F plus N-acetylneuraminic acid (+291 Da) will have a similar mass as G1F/G1F plus Lysine (+162+128=290 Da). However, the addition of sialic acidic will have an acidic shift on pI, while unprocessed C-terminal lysine has a basic pI shift. With the charge separation in the front end before MS detection, these two proteoforms with similar mass can be spatially separated and confidently identified according to the charge shift.

Another advantage of icIEF-UV/MS analysis is the ability to identify glycation events without deglycosylation treatment. Glycation has a mass shift of 162 Da which is the same as an addition of galactose in glycosylation. Glycosylation is a neutral reaction where different glycosylation pairs have the same charge, while the addition of hexose will cause an acidic shift. With the advantage of front-end icIEF charge separation, if the relative intensity of higher glycosylation pairs is increasing along with the acidic shift, it indicates the glycation events in the molecule.

The icIEF-UV/MS analysis on the subunit level provides the domain information of the PTMs that are detected on the intact level. It can also potentially simplify the analysis by looking at each region of the molecule separately with fewer PTMs, compared to all the PTMs, to look at the intact level as a whole.

icIEF-UV/MS can be used at different stages during development, from cell line development to late phase characterization, whenever there is a need to identify the charge variants. For example, at an early stage, scientists could use this technology for product comparison between different cell lines and quickly identify the differences between them. At a later phase, this technology could be utilized for forced degradation studies to identify each charge variant observed from icIEF. This technology can also be used to compare different biosimilars or processing batches.