Modeling of bispecific antibody elution in mixed-mode cation-exchange chromatography.

The increasing demand for cost-efficient manufacturing of biopharmaceuticals has been the main driving force for the development of novel chromatography resins, which resulted in the development of multimodal or mixed-mode chromatographic resins. Most of them combine electrostatic and hydrophobic functionalities and are designed to deliver unique selectivity and increased binding capacities also at increased ionic strength. However, the mechanism of the protein-resin interaction in mixed-mode chromatography is still not fully understood. The performance of protein separations in mixed-mode chromatography is consequently difficult to predict. In this work, we present a model combining both salt and pH dependence to characterize and to predict protein retention in mixed-mode chromatography. The model parameters are determined based on simple linear pH gradient elution experiments at different ionic strengths and they are directly transferable for the prediction of salt-induced elution at fixed pH. Validity of the model is demonstrated for a bispecific antibody and its product-related impurities.

Anti-Langmuir elution behavior of a bispecific monoclonal antibody in cation exchange chromatography: Mechanistic modeling using a pH-dependent Self-Association Steric Mass Action isotherm.

The objective of this scientific work was to model and simulate the complex anti-Langmuir elution behavior of a bispecific monoclonal antibody (bsAb) under high loading conditions on the strong cation exchange resin POROS™ XS. The bsAb exhibited anti-Langmuirian elution behavior as a consequence of self-association expressed both in uncommon retentions and peak shapes highly atypical for antibodies. The widely applied Steric Mass Action (SMA) model was unsuitable here because it can only describe Langmuirian elution behavior and is not able to describe protein-protein interactions in the form of self-association. For this reason, a Self-Association SMA (SAS-SMA) model was applied, which was extended by two activity coefficients for the salt and protein in solution. This model is able to describe protein-protein interactions in the form of self-dimerization and thus can describe anti-Langmuir elution behavior. Linear gradient elution (LGE) experiments were carried out to obtain a broad dataset ranging from pH 4.5 to 7.3 and from 50 to 375 mmol/L Na+ for model parameter determination. High loading LGE experiments were conducted with an increasing load from 0.5 up to 75.0 mgbsAb/mLresin. Thereby, pH-dependent empirical correlations for the activity coefficient of the solute protein, for the equilibrium constant of the self-dimerization process and for the shielding factor could be set up and ultimately incorporated into the SAS-SMA model. This pH-dependent SAS-SMA model was thus able to simulate anti-Langmuir behavior over extended ranges of pH, counterion concentration, and column loading. The model was confirmed by experimental verification of simulated linear pH gradient elutions up to a load of 75.0 mgbsAb/mLresin.

End-to-end approach for the characterization and control of product-related impurities in T cell bispecific antibody preparations.

Antibody-based T cell-activating biologics are promising therapeutic medicines being developed for a number of indications, mainly in the oncology field. Among those, T cell bispecific antibodies are designed to bind one tumor-specific antigen and the T cell receptor at the same time, leading to a robust T cell response against the tumor. Although their unique format and the versatility of the CrossMab technology allows for the generation of safer molecules in an efficient manner, product-related variants cannot be completely avoided. Therefore, it is of extreme importance that both a manufacturing process that limits or depletes product-related impurities, as well as a thorough analytical characterization are in place, starting from the development of the manufacturing cell line until the assessment of potential toxicities. Here, we describe such an end-to-end approach to minimize, quantify and control impurities and -upon their functional characterization- derive specifications that allow for the release of clinical material.

Mechanism of improved antibody aggregate separation in polyethylene glycol-modulated cation exchange chromatography.

Ion-exchange chromatography is used in biopharmaceutical downstream processes to reduce product-related impurity levels. Because protein aggregate levels can be considered as a critical quality attribute, the removal of aggregated protein species is of primary importance. The addition of polyethylene glycol (PEG) to the mobile phase in ion-exchange chromatography was found to significantly improve the chromatographic separation of monomers from aggregates. In this work, linear gradient elution experiments with monomeric and aggregated samples of a monoclonal antibody were performed on a strong cation exchange resin at different PEG concentrations to investigate the underlying effects responsible for the observed selectivity improvement. PEG is well known to be excluded from a surface layer volume around the protein and the stationary phase; thus, enhancing adsorption of the preferentially hydrated protein to the hydrated stationary phase. The exclusion volume depends on the accessible surface area of the protein leading to a stronger influence of PEG on larger protein species and thus an improved separation of monomer and aggregates. This hypothesis could be consolidated comparing the distribution equilibrium in PEG solution to that in water by calculating equilibrium constants and transfer free energies using the chromatographic data from the linear gradient elution experiments performed at different pH values.

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 2. Investigation of high loading and column overloading effects.

The application of a model-based approach for industrial chromatography development requires the capability of the model to describe protein elution under high loading and overloading conditions. In a previous work, an extensive dataset was created to model the elution behavior of a bispecific antibody (bsAb) on the strong cation exchange resin POROS™ XS. Thereby, the pH-dependence of the model parameters in the Steric Mass Action (SMA) model could be examined and described over a pH range of 4.5 to 8.9. However, discrepancies between simulated and experimental data were observed under high loading and overloading conditions, particularly in the lower pH range (pH 4.5 to 5.3) and in the higher pH range (pH 6.0 to 9.0). In this work, these discrepancies are studied by performing new experiments which show that these differences were primarily not caused by limitations of the SMA model. At lower pH values, overloading phenomena such as protein breakthrough during the loading phase, additional peaks, and peak shoulders occurred. The application of various experiments performed with different Na+ concentrations and different loading times during sample loading revealed that intraparticle diffusion effects and conformational changes of the bsAb are responsible for these overloading phenomena at low pH. The applied lumped rate mass transfer model is not adequate and should be extended to consider these effects. At higher pH, the assumption of describing the bsAb's elution behavior with only one simulated species was insufficient to predict complex peak shapes that arise because of multi-component elution of the bsAb's charge variants. The extension of the model to a simple multi-component system consisting of two variants allowed the prediction of a majority of the complex elution profiles.

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 1. Investigation of the influence of pH on the steric shielding factor.

In ion exchange chromatography, the Steric Mass Action (SMA) formalism is frequently used to simulate sorption processes at low and high column load conditions. To apply the SMA model for describing protein elution over wide ranges of pH, it is necessary to use pH-dependent model parameters. In the past, some publications have already described the pH-dependence of the characteristic protein charge and the equilibrium constant, while the influence of pH on the steric shielding factor has been mostly neglected. In this work, the pH-dependences of all relevant model parameters, including the shielding factor, were investigated, described, and implemented into the SMA model. Therefore, the elution behavior of a bispecific monoclonal antibody on the strong cation exchange resin POROS™ XS was modeled over broad ranges of pH, salt concentrations, and protein concentrations. Linear gradient elution experiments were performed to generate an extensive data set by using increasing column loadings from 0.5 up to 75.0 mgbsAb/mLresin. By using an inverse peak fitting method, shielding factors were estimated at various pH values ranging from 4.5 to 8.9. The results showed that an increasing buffer pH resulted in strongly increasing shielding factors. A semi-empirical correlation describing the shielding factor as a function of pH was established and implemented into the SMA formalism. This approach led to precise prediction of protein elution behavior using a single-component simulation. This was demonstrated by accurate simulation of linear salt, pH and dual gradient elution experiments conducted under high loading conditions.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors.

PURPOSE: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. EXPERIMENTAL DESIGN: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. RESULTS: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA-expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1-positive tumors. CONCLUSIONS: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286-97. ©2016 AACR.

Solvent modulation strategy for superior antibody monomer/aggregate separation in cation exchange chromatography.

Cation exchange chromatography (CEX) is an integral part of many downstream processes for monoclonal antibodies (mAbs). However, in some cases CEX methods with standard mobile phase conditions do not lead to a sufficient removal of soluble antibody aggregates. The addition of neutral polymers such as polyethylene glycol (PEG) to the mobile phase can improve the separation of proteins in IEC remarkably. The applicability of this solvent modulation technique is limited by protein precipitation at higher PEG concentrations. To overcome this limitation solubility enhancers like polyols and amino acids can be added to the mobile phase. These additives are known to inhibit PEG-induced protein precipitation in solution. This new solvent modulation strategy was tested with three different mAbs on two different CEX resins in the presence of PEG in combination with various solubility enhancers. In order to assess the general applicability of this method, mAbs were selected that show major differences with respect to their sensitivity to PEG-induced precipitation and monomer/aggregate resolution performance that is achieved by CEX under standard conditions. For all three mAbs precipitation could be prevented without elimination of the positive PEG-effect. The addition of solubility enhancers gives access to improved separation at elevated PEG concentrations and high protein loadings without running into precipitation issues. Our data indicate that this method is generically applicable and leads to a superior antibody monomer/aggregate separation.

Solvent modulated linear pH gradient elution for the purification of conventional and bispecific antibodies: Modeling and application.

Classical ion-exchange chromatography using a linear salt gradient to elute the adsorbed protein at fixed pH is the most common method to separate product-related impurities during downstream processing of biopharmaceuticals. Linear pH gradient elution provides a useful alternative by separating proteins in a linear pH gradient at fixed salt concentration. Although linear pH gradient elution provides excellent selectivity, it is rarely encountered in industrial purification processes. Here, a stoichiometric displacement model is used to characterize pH gradient elution based on simple linear gradient elution experiments. Protein retention behavior is described with respect to the pH dependencies of the characteristic binding charge and the equilibrium constant of the ion exchange reaction. Furthermore, the influence of solvent composition using PEG as a mobile phase modifier is investigated. Validity and applicability of the model are demonstrated for the purification of a conventional monoclonal antibody from soluble aggregates and for a novel bispecific antibody format containing a unique product-related impurity profile. pH step elution protocols are derived from model calculations without further optimization experiments necessary.