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The identification and quantification of post-translational modifications (PTMs) is a crucial step required during the development of therapeutic proteins. In particular, the characterization of charge variants separated by cation exchange chromatography (CEX) is a tedious process commonly performed with an off-line manual fraction collection followed by peptide mapping. To improve the efficiency of this time-consuming approach, a generic on-line multi-dimensional LC/MS approach was developed for the characterization of various monoclonal antibody (mAb) isotypes and a bi-specific antibody (BsAb). Fractions collected from 1D CEX analysis were consecutively reduced on a 2D reversed phase liquid chromatography (RPLC) column (polyphenyl), digested within 1–2 min using a 3D immobilized trypsin cartridge, and finally the obtained peptides were separated on another 4D RPLC column (C18), and simultaneously identified with a Q Exactive™ mass spectrometer. 2D RPLC columns and 3D trypsin cartridges from different suppliers were compared, as well as the effects of reducing agents. The effect of 2D and 4D RPLC column temperature, and 2D RPLC column mass load were also systematically studied. Under optimal conditions, the multi-dimensional LC/MS system described in this paper is a robust tool for the on-line digestion of proteins and shows high repeatability. Similar levels of oxidation and deamidation were measured using the off-line and on-line approaches for the same stressed samples. The lower amounts of deamidation and isomerization measured at some asparagine and aspartic acid residues by the on-line approach compared to the manual off-line procedure suggest reduced artifacts using the on-line methodology. The multi-dimensional LC/MS described here enables fast, on-line, automated characterization of therapeutic antibodies without the need for off-line fraction collection and sample pre-treatment (manual approach). The entire workflow can be completed within less than one day, compared to weeks with the manual off-line procedure.

Inhibition of the interleukin-2 (IL-2) pathway has potent immunosuppressive activity in humans as is evident from the broad therapeutic utility of cyclosporine, rapamycin, tacrolimus, and monoclonal antibodies blocking the high-affinity subunit of the IL-2 receptor (CD25). Here we describe a humanized antibody, MT204, interfering with IL-2 signaling by a novel mechanism. Although MT204 did not prevent IL-2 from binding to CD25, it potently antagonized downstream signaling events of IL-2 at sub-nanomolar concentrations, such as STAT3 tyrosine phosphorylation, expression of CD124, production of gamma-interferon and cell proliferation. While MT204 and the anti-CD25 mAb daclizumab were equally effective in inhibiting autocrine growth of human CD4(+) T cells, MT204 was far superior in preventing proliferation of NKL lymphoma cells, production of gamma-interferon by natural killer (NK) cells and proliferation of primary NK cells. MT204 has potential as a novel immunosuppressive and anti-proliferative therapy with an apparently broader spectrum of activities than anti-CD25 antibodies.

Many kinds of bispecific antibodies recruiting T cells for cancer therapy have been developed. Side-by-side comparison has shown that CD19-/CD3-bispecific antibodies of the diabody, tandem diabody (Tandab) and quadroma format had similar cytotoxic activity, with Tandab being the most active format. Tandab has also been claimed to be superior to single-chain (sc) Fv-based bispecific constructs although data from a side-by-side comparison are not available. In this study, we compared side-by-side MT103 (bscCD19xCD3), a single-chain bispecific antibody of the BiTE class, with a CD19-/CD3-bispecific representative of the Tandab class. Based on literature data, we have constructed, produced and characterized the LL linker version of Tandab, which was reported to be the most active version of Tandab proteins. A dimeric protein of 114kDa was obtained that showed proper bispecific binding to CD3- and CD19-positive cells and could redirect both pre-stimulated and unstimulated human T cells for lysis of human B lymphoma lines Raji, MEC-1 and Nalm-6. Raji cells were lysed at a half-maximal concentration (EC50) of 10 nM Tandab using pre-stimulated T cells, which closely matched the published activity of LL-Tandab with this particular cell line. MT103 had between 700- and 8000-fold higher efficacy than Tandab for redirected lysis of the three human B lymphoma lines. These data demonstrate that under identical experimental conditions, the BiTE format has far superior activity compared to the Tandab format and is also superior to conventional diabody and quadroma formats. The extraordinary potency of the BiTE class and its representative MT103 may translate into improved anti-tumor activity, lower dosing and lower costs of production compared to other bispecific antibody formats.