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With respect to all biopharmaceuticals marketed to date, monoclonal antibodies represent the largest fraction with more than 48% market share (2012). However, the development of biopharmaceutical formulations is a challenging task, and time-consuming and cost-intensive high-throughput screenings are still state-of-the-art in formulation design. These screening techniques are almost exclusively based on heuristic decisions thus the benefit in terms of mechanistic understanding is often unclear. It requires novel, physical-sound methods to enhance/optimize future formulation development, ideally by understanding molecular interactions in these complex solutions. A suitable and evaluated measure-of-choice to characterize protein-protein interactions in aqueous protein solutions is the second osmotic virial coefficient B 22 which can be measured using static light scattering techniques. Furthermore B 22 can be modeled/predicted via the extended mxDLVO model for protein-protein interactions in the presence of single excipients and excipient-mixtures. Building up on this approach, giving an additional insight into water-water and water-excipient interactions, the thermodynamic equation-of-state ePC-SAFT is used to calculate water activity coefficients in the presence of excipient-mixtures. Immunoglobulin G (IgG) was chosen as a model protein to predict B 22 -values for IgG in the presence of model excipient-mixtures (trehalose-NaCl, l-histidine-trehalose, l-histidine-NaCl). The combination of water activity coefficients and B 22 allows to quickly identify a first guess on suitable formulation conditions that then can be further evaluated with existing methods/knowledge., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aqueous two-phase extraction (ATPE) of therapeutic proteins is a promising separation alternative to cost-intensive chromatography, still being the workhorse of nowadays downstream processing. As shown in many publications, using NaCl as displacement agent in salt-polymer ATPE allows for a selective purification of the target protein immunoglobulin G (IgG) from human serum albumin (HSA, represents the impurity). However a high yield of the target protein is only achievable as long as the protein is stabilized in solution and not precipitated. In this work the combined influence of NaCl and polyethylene glycol (Mw=2000g/mol) on the IgG-IgG interactions was determined using composition gradient multi-angle light scattering (CG-MALS) demonstrating that NaCl induces a solubilization of IgG in polyethylene glycol 2000 solution. Moreover it is shown that the displacement agent NaCl has a significant and beneficial influence on the IgG solubility in polyethyleneglycol2000-citrate aqueous two-phase system (ATPS) which can also be accessed by these advanced B 22 measurements. By simultaneous consideration of IgG solubility data with results of the ATPS phase behavior (especially volume fraction of the respective phases) allows for the selection of process tailored ATPS including identification of the maximum protein feed concentration. Through this approach an ATPS optimization is accessible providing high yields and selectivity of the target protein (IgG)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The purification of therapeutic proteins is a challenging task with immediate need for optimization. Besides other techniques, aqueous 2-phase extraction (ATPE) of proteins has been shown to be a promising alternative to cost-intensive state-of-the-art chromatographic protein purification. Most likely, to enable a selective extraction, protein partitioning has to be influenced using a displacement agent to isolate the target protein from the impurities. In this work, a new displacement agent (lithium bromide [LiBr]) allowing for the selective separation of the target protein IgG from human serum albumin (represents the impurity) within a citrate-polyethylene glycol (PEG) ATPS is presented. In order to characterize the displacement suitability of LiBr on IgG, the mutual influence of LiBr and the phase formers on the aqueous 2-phase system (ATPS) and partitioning is investigated. Using osmotic virial coefficients (B22 and B23) accessible by composition gradient multiangle light-scattering measurements, the precipitating effect of LiBr on both proteins and an estimation of both protein partition coefficients is estimated. The stabilizing effect of LiBr on both proteins was estimated based on B22 and experimentally validated within the citrate-PEG ATPS. Our approach contributes to an efficient implementation of ATPE within the downstream processing development of therapeutic proteins., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

A promising alternative to state of the art chromatographic separations of therapeutic proteins is the extraction of the target protein using an aqueous two-phase system (ATPS). The use of an additional salt working as a displacement agent can influence the protein partitioning behavior in ATPS and thus enable a selective purification of the target protein. The selection of a suitable ATPS for protein extraction requires information concerning the protein-protein interactions (second osmotic virial coefficient B22 ) as well as the interactions between protein and solute (displacement agent and phase-forming components) (cross virial coefficient B23 ). In this work, the partitioning behavior and the precipitation affinity of immunoglobulin G (IgG) is considered within a polyethylene glycol (PEG)-phosphate ATPS. The influence on IgG partitioning upon addition of NaCl and (NH4)2 SO4 was investigated. In order to access the IgG precipitation affinity and the IgG partitioning behavior, the B22 and B23 values were determined for several combinations of solute [PEG, phosphate buffer, NaCl, and (NH4)2 SO4 ] and IgG based on static light scattering measurements. A qualitative estimation of the IgG precipitation affinity and the suitability of a solute as potential displacement agent within the PEG-phosphate ATPS on the basis of the measured B22 and B23 values is presented., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The extraction of therapeutic proteins like monoclonal antibodies in aqueous two-phase systems (ATPS) is a suitable alternative to common cost intensive chromatographic purification steps within the downstream processing. Thereby the protein partitioning can be selectively changed using a displacement agent (additional salt) in order to allow for a successful purification of the target protein. Within this work a new shortcut strategy for the calculation of protein partition coefficients in polymer-salt ATPS is presented. The required protein-solute (phase-forming component, displacement agent) interactions are covered by the cross virial coefficient B 23 measured by composition gradient multi-angle light scattering (CG-MALS). Using this shortcut calculation allows for an efficient determination of the partition coefficients of the target protein immunoglobulin G ( IgG ) and the impurity human serum albumin ( HSA ) within PEG-citrate and PEG-phosphate ATPS independently on the protein concentration. We demonstrate that the selection of a suitable displacement agent allowing for a selective purification of IgG from HSA is accessible by B 23 . Based on the determination of the protein–protein interactions via CG-MALS covered by the second osmotic virial coefficient B 22 a further optimization of ATPS preventing protein precipitation is enabled. The results show that our approach contributes to an efficient downstream processing development. [ABSTRACT FROM AUTHOR]

Within the last decade, thermodynamic models have increasingly grown into the pharmaceutical industry, becoming an essential part in both, process development, as well as formulation development of small-molecule drugs. They are to an increasing degree accepted as a valuable building block to evaluate e.g. small-molecule formulation conditions. In contrast, modeling of highly complex (bio)-molecules (e.g. monoclonal antibodies) remains a challenging task and thermodynamic modeling is only rarely applied for e.g. protein formulations in the biopharmaceutical industry. Within this work, we developed a thermodynamic modeling approach to access protein-protein and protein-excipient interactions (via second osmotic virial coefficients B 22 and cross viral coefficients B 23 ) for immunoglobulin G (IgG) and bovine serum albumin (BSA) in the presence of different excipients (NaCl, sucrose, L-histidine, L-arginine) at defined buffer conditions using the e PC-SAFT equation-of-state. The pure component parameters of IgG were determined based on the amino acid sequence and further fitted to experimentally determined static light scattering signals of IgG at buffer conditions. This approach allows to predict protein-protein and protein-excipient interactions of BSA and IgG in the presence of different excipient types and thus give access to protein phase behavior in these complex solutions. Applying this approach further allows to gain a mechanistic understanding on the complex interactions in solution that govern the phase behavior, and thus can aid an optimized formulation development. • Modeling protein-protein interactions using the e PC-SAFT equation-of-state. • Novel modeling strategy for second osmotic and cross virial coefficients for proteins in the presence of excipients. • Predicting protein-protein/protein-excipient interactions in the presence of excipients based on pure component parameters. • Access to other thermodynamic quantities such as solution densities. [ABSTRACT FROM AUTHOR]

Early- and late-stage formulation development for biopharmaceuticals (e.g. therapeutic proteins) remains a challenging task, especially when one has to select suitable excipients to solubilize and stabilize the protein in solution. State-of-the-art formulation development includes cost-intensive high-throughput screening methods to identify suitable excipients and formulation conditions. These methods often deliver a "working" formulation but unlikely the "optimal" formulation. Within this work, we developed a novel method that allows identifying suitable excipients based on the second osmotic virial coefficient B 22 as the measure of choice for protein-protein interactions in aqueous solution in the presence of excipients (salts, sugars, amino acids, etc.). B 22 is easily accessible by light scattering methods and advanced thermodynamic models like the extended mxDLVO model. Aqueous immunoglobulin G solutions including sugars, amino acids, surfactants and salts as excipients were used as model systems within this work. Applying the extended mxDLVO, the model allows predicting protein-protein interactions with high accuracy for mixtures of up to three excipients in one formulation. These measurements allow for quick identification of suitable excipients and their concentrations and are giving a greater insight in molecular interactions under the respective formulation conditions. An application of this method in future formulation development has the potential to reduce time-consuming and cost-intensive screening methods and finally lead to optimal formulations. • Existing mxDLVO model was modified to applied to protein formulations. • Modeling of second osmotic virial coefficients in presence of excipients. • Prediction of second osmotic virial coefficients in presence of excipient-mixtures. • Application of this model avoids time-consuming formulation screening. [ABSTRACT FROM AUTHOR]

• Optimized ATPS extraction performance for ATPE of biomolecules through excipients. • Rapid identification of suitable excipients by methodical selection. • Improved conformational and colloidal biomolecule stability. • Decreased biomolecule precipitation and increased yield through improved stability. • Simplified process handling by biomolecule stability and solubility improvements Despite the benefits of aqueous two-phase extraction (ATPE) for purification of high-value biomolecules, phase-formers that are used to generate the aqueous two-phase system (ATPS) often reduce the stability and solubility of the biomolecule. Within this work, we thus evaluated the use of suitable excipients to increase the biomolecule stability and solubility during ATPE, whilst not negatively affecting yield and selectivity. Applying this new physically sound approach enabled a rapid selection of L-arginine as suitable excipient for the ATPE of Immunoglobulin G. It was demonstrated that both the (conformational and colloidal) stability and solubility of Immunoglobulin G in the ATPS were increased, thus maximizing yield, minimizing precipitation, and improving the extraction performance of the ATPS. The applicability and transferability of the approach was confirmed by optimizing the ATPE of an industrial relevant amine dehydrogenase. The optimized ATPS enabled an extraction yield of 79.17 wt% (impurities < 7 wt%) directly from crude extract. Furthermore, using the excipients broadened the ATPE process window, thus simplifying process handling during ATPE and further downstream unit operations. Prospectively, the use of excipients can support the establishment of ATPE as downstream technology. [Display omitted] [ABSTRACT FROM AUTHOR]