Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer.

The increasing importance of immunoglobulins G (IgGs) as biotherapeutics calls for improved structural characterization methods designed for these large (~150kDa) macromolecules. Analysis workflows have to be rapid, robust, and require minimal sample preparation. In a previous work we showed the potential of Orbitrap Fourier transform mass spectrometry (FTMS) combined with electron transfer dissociation (ETD) for the top-down investigation of an intact IgG1, resulting in ~30% sequence coverage. Here, we describe a top-down analysis of two IgGs1 (adalimumab and trastuzumab) and one IgG2 (panitumumab) performed with ETD on a mass spectrometer equipped with a high-field Orbitrap mass analyzer. For the IgGs1, sequence coverage comparable to the previous results was achieved in a two-fold reduced number of summed transients, which corresponds, taken together with the significantly increased spectra acquisition rate, to ~six-fold improvement in analysis time. Furthermore, we studied the influence of ion-ion interaction times on ETD product ions for IgGs1, and the differences in fragmentation behavior between IgGs1 and IgG2, which present structural differences. Overall, these results reinforce the hypothesis that gas phase dissociation using both energy threshold-based and radical-driven ion activations is directed to specific regions of the polypeptide chains mostly by the location of disulfide bonds. SIGNIFICANCE OF THE STUDY: Compared with our previous report, the results presented herein demonstrate the power of technological advances of the next generation Orbitrap™ platform, including the use of a high-field compact (i.e., D20) Orbitrap mass analyzer, and a dedicated manipulation strategy for large protein ions (via their trapping in the HCD collision cell along with reduction of the pressure in the cell). Notably, these important developments became recently commercially available in the top-end Orbitrap platforms under the name of "Protein Mode". Furthermore, we continued exploring the advantages offered by the summation (averaging) of transients (time-domain data) for improving the signal-to-noise ratio of top-down mass spectra. Finally, for the first time we report the application of the hybrid ion activation technique that combines electron transfer dissociation and higher energy collisional dissociation, known as EThcD, on intact monoclonal antibodies. Under these specific instrumental parameters, EThcD produces a partially complementary fragmentation pattern compared to ETD, increasing the overall sequence coverage especially at the protein termini.

Characterization of the N-terminal heterogeneities of monoclonal antibodies using in-gel charge derivatization of α-amines and LC-MS/MS.

The bioproduction of recombinant monoclonal antibodies results in complex mixtures of a main isoform and numerous macro- and microvariants. Monoclonal antibodies therefore present different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities, the truncation and fragmentation of the primary amino-acid sequence result in shorter or cleaved polypeptide chains while the incomplete processing of the signal peptide produces N-terminal elongated polypeptide chains. Here, we present an in-gel protein N-terminal chemical derivatization method using (N-succinimidyloxycarbonylmethyl)-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This chemical tag enhances the detection by mass spectrometry of the N-terminal positions of proteins and allows their unambiguous assignment without altering the identification of internal digestion peptides. This method adds just one step to the classical peptide mapping workflow. Using this in-gel N-TOP (N-terminal oriented proteomics) strategy, the N-terminal sequence heterogeneities of several monoclonal antibodies, among which are minor unexpected proteoforms, were successfully detected and characterized.

Innovative native MS methodologies for antibody drug conjugate characterization: High resolution native MS and IM-MS for average DAR and DAR distribution assessment.

Antibody drug conjugates (ADCs) are macromolecules composed of cytotoxic drugs covalently attached via a conditionally stable linker to monoclonal antibodies (mAbs). ADCs are among the most promising next generation of empowered mAbs foreseen to treat cancers. Compared to naked mAbs, ADCs have an increased level of complexity as the heterogeneity of conjugation cumulates with the inherent microvariability of the biomolecule. An increasing need underlying ADC's development and optimization is to improve its analytical and bioanalytical characterization by assessing three main ADC quality attributes: drug distribution, amount of naked antibody, and average drug to antibody ratio (DAR). Here, the analytical potential of native mass spectrometry (MS) and native ion mobility MS (IM-MS) is compared to hydrophobic interaction chromatography (HIC), the reference method for quality control of interchain cysteinyl-linked ADCs. Brentuximab vedotin, first in class and gold standard, was chosen for a proof of principle. High resolution native MS provided accurate mass measurement (<30 ppm) of intact ADCs together with average DAR and drug distribution, confirming the unique ability of native MS for simultaneous detection of mixtures of covalent and noncovalent products. Native IM-MS was next used for the first time to characterize an ADC. IM-MS evidenced ADC multiple drug loading, collisional cross sections measurement of each payload species attesting slight conformational changes. A semiquantitative interpretation of IM-MS data was developed to directly extrapolate average DAR and DAR distribution. Additionally, HIC fractions were collected and analyzed by native MS and IM-MS, assessing the interpretation of each HIC peak. Altogether, our results illustrate how native MS and IM-MS can rapidly assess ADC structural heterogeneity and how easily these methods can be implemented into MS workflows for in-depth ADC analytical characterization.

Advantages of extended bottom-up proteomics using Sap9 for analysis of monoclonal antibodies.

Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion.

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA )-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms,electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glycoprofiling and demonstration of the absence of additional conjugation was easily achieved. As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis.

The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers' yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations and RNA interference-mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammonium stress.