Purification of hydrophobic complex antibody formats using a moderately hydrophobic mixed mode cation exchange resin.

Immunoglobulins of complex formats possess great potential for increased biopharmaceutical efficacy. However, challenges arise during their purification as the removal of numerous product-related impurities typically requires several expensive chromatographic steps. Additionally, many complex antibody formats have a high hydrophobicity which impairs the use of conventional mixed mode chromatography. In the present study, both of these challenges were addressed through the development of an innovative mixed mode resin with 2-amino-4methylpentanoic acid ligands that combines weak cation exchange with moderate hydrophobic interactions. Supported by high throughput partition coefficient screens for identification of preferable pH and salt concentration ranges in bind and elute mode, this mixed mode resin successfully demonstrated efficient impurity separation from an extremely hydrophobic bispecific antibody with a single unit operation. High purity (>97%) was obtained as a result of significant reduction of product-related impurities as well as process-related host cell proteins (>3 log scale), while maintaining satisfactory recovery (70%). This also supports that highly hydrophobic antibody formats can be efficiently purified using a resin with moderate hydrophobic characteristics. Studies involving additional antibodies possessing different formats and a wide range of hydrophobicity confirmed the broad applicability of the new resin. In view of its high selectivity and robust operating ranges, as well as the elimination of the need for an additional column step, the novel resin enables simplified downstream processing and economic manufacturing of complex antibody formats.

Development of versatile affinity-based system for one step purification process: Case of Group A Streptococcus vaccine.

Affinity capture is one of the most attractive strategies for simplifying downstream processing. Although it is a key mainstream approach for antibody purification, the same is not true for other biologics such as vaccines, mainly due to the lack of suitable affinity material. In this study, a novel custom affinity system is introduced permitting widespread adoption of affinity capture for the purification of biologics beyond antibodies. This is illustrated here by the development of a one-step purification process of a mutant form of streptolysin O (SLO), a vaccine candidate against Streptococcus pyogenes infection. The system consists of the association of custom ligands based on the Nanofitin protein scaffold, with Eshmuno® industry-grade chromatography medium. The Nanofitins were selected for their specificity to the target product. The newly developed affinity medium was used at different column sizes to monitor scalability from process development (1 ml) and robustness verification (5 ml) to pilot (133 ml) and technical (469 ml) runs. The single-step affinity purification consistently delivered high purity product (above > 90%) and improved performances compared with the current three-step process: reduced process time and footprint (3 to 1 step) and increased product yields (0.31 g vs. 0.04 g of SLO per kg of harvest broth). The custom affinity system herein described can potentially be applied to any biologic for which a specific Nanofitin is identified, thus establishing a platform with a strong impact on the manufacturing of vaccines and other biological targets.

Influence of excipients in Protein A chromatography and virus inactivation.

The purification of monoclonal antibodies and Fc fusion proteins consist of several unit operations operated commonly as a platform approach, starting with Protein A chromatography. The first capture step, the following low pH virus inactivation, and subsequent ion exchange chromatography steps are mostly able to remove any impurities, like host cell proteins, aggregates, and viruses. The changes in pH and conductivity during these steps can lead to additional unwanted product species like aggregates. In this study, excipients with stabilizing abilities, like polyols, were used as buffer system additives to study their impact on several aspects during Protein A chromatography, low pH virus inactivation, and cation exchange chromatography. The results show that excipients, like PEG4000, influence antibody elution behavior, as well as host-cell protein elution behavior in a pH-gradient setup. Sugar excipients, like Sucrose, stabilize the antibody during low pH virus inactivation. All excipients tested show no negative impact on virus inactivation and dynamic binding capacity in a subsequent cation exchange chromatography step. This study indicates that excipients and, possibly excipient combinations, can have a beneficial effect on purification without harming subsequent downstream processing steps.

Mid-infrared spectroscopy-based analysis of mammalian cell culture parameters.

Within the framework of process analytical technology, infrared spectroscopy (IR) has been used for characterization of biopharmaceutical production processes. Although noninvasive attenuated total reflection (ATR) spectroscopy can be regarded as gold standard within IR-based process analytics, simpler and more cost-effective mid-infrared (MIR) instruments might improve acceptability of this technique for high-level monitoring of small scale experiments as well as for academia where financial restraints impede the use of costly equipment. A simple and straightforward at-line mid-IR instrument was used to monitor cell viability parameters, activity of lactate dehydrogenase (LDH), amount of secreted antibody, and concentration of glutamate and lactate in a Chinese hamster ovary cell culture process, applying multivariate prediction models, including only 25-28 calibration samples per model. Glutamate amount could be predicted with high accuracy (R(2) 0.91 for independent test-set) while antibody concentration achieved good prediction for concentrations >0.4 mg L(-1) . Prediction of LDH activity was accurate except for the low activity regime. The model for lactate monitoring was only moderately good and requires improvements. Relative cell viability between 20 and 95% could be predicted with low error (8.82%) in comparison to reference methods. An initial model for determining the number of nonviable cells displayed only acceptable accuracy and requires further improvement. In contrast, monitoring of viable cell number showed better accuracy than previously published ATR-based results. These results prove the principal suitability of less sophisticated MIR instruments to monitor multiple parameters in biopharmaceutical production with relatively low investments and rather fast calibration procedures.

Development of a univariate membrane-based mid-infrared method for protein quantitation and total lipid content analysis of biological samples.

Biological samples present a range of complexities from homogeneous purified protein to multicomponent mixtures. Accurate qualification of such samples is paramount to downstream applications. We describe the development of an MIR spectroscopy-based analytical method offering simultaneous protein quantitation (0.25-5 mg/mL) and analysis of total lipid or detergent species, as well as the identification of other biomolecules present in biological samples. The method utilizes a hydrophilic PTFE membrane engineered for presentation of aqueous samples in a dried format compatible with fast infrared analysis. Unlike classical quantification techniques, the reported method is amino acid sequence independent and thus applicable to complex samples of unknown composition. By comparison to existing platforms, this MIR-based method enables direct quantification using minimal sample volume (2 µL); it is well-suited where repeat access and limited sample size are critical parameters. Further, accurate results can be derived without specialized training or knowledge of IR spectroscopy. Overall, the simplified application and analysis system provides a more cost-effective alternative to high-throughput IR systems for research laboratories with minimal throughput demands. In summary, the MIR-based system provides a viable alternative to current protein quantitation methods; it also uniquely offers simultaneous qualification of other components, notably lipids and detergents.

Feasibility of polyelectrolyte-driven Fab fragment separation.

The use of antigen-binding fragments (Fabs) as biotherapeutic agents is gaining interest and thus requires development of adequate purification strategies aimed at separating Fabs from other proteins. Thus, the feasibility of using a copolymer for separation of Fabs from monoclonal antibodies (mAbs) and fragment constant regions (Fcs) was evaluated, employing a blend of purified solutions of these proteins. The use of a copolymer exerting both hydrophobic as well as anionic properties resulted in high precipitation yields for both the mAb and Fc fragment, even at ionic strength of 150 mM NaCl. On the contrary, Fabs exhibited reduced precipitation yields upon copolymer addition. These observations are attributed to differences in protein physicochemical parameters, allowing mAbs and Fcs to be precipitated via conjoint electrostatic and hydrophobic interactions. In contrast, Fabs were mainly precipitated via electrostatic interactions, being reduced at higher ionic strength. This finding was corroborated by hydrophobicity analysis using 2-p-toluidinonaphthalene-6-sulfonate, showing enhanced hydrophobicity of Fcs compared to mAbs alone, while Fabs exhibited the lowest hydrophobicity. Within the context of increasing demand for Fabs as therapeutic proteins, these results may open up a simpler purification strategy for this protein class, potentially also to be implemented within the context of polymer-driven protein purification during fermentation.

Customization of copolymers to optimize selectivity and yield in polymer-driven antibody purification processes.

This manuscript describes customization of copolymers to be used for polymer-driven protein purification in bioprocessing. To understand how copolymer customization can be used for fine-tuning, precipitation behavior was analyzed for five target antibodies (mAbs) and BSA as model impurity protein, at ionic strength similar to undiluted cell culture fluid. In contrast to the use of standardized homopolymers, customized copolymers, composed of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and 4-(acryloylamino)benzoic acid (ABZ), exhibited antibody precipitation yields exceeding 90%. Additionally, copolymer average molecular weight (Mw ) was varied and its influence on precipitation yield and contaminant coprecipitation was investigated. Results revealed copolymer composition as the major driving force for precipitation selectivity, which was also dependent on protein hydrophobicity. By adjusting ABZ content and Mw of the precipitant for each of the mAbs, conditions were found that allowed for high precipitation yield and selectivity. These findings may open up new avenues for using polymers in antibody purification processes.

Mid-infrared spectroscopy-based antibody aggregate quantification in cell culture fluids.

Therapeutic antibody purification involves several steps which potentially induce antibody aggregation. Currently, aggregate monitoring mainly employs chromatographic, SDS-PAGE and light scattering techniques. In this study, the feasibility of mid-infrared spectroscopy (MIR) for the quantification of soluble antibody aggregates was investigated. Several multivariate models were evaluated to quantify antibody aggregation in chromatography elution streams and in clarified CHO cell culture supernatants (a surrogate for bioreactor output). A general model was established that is applicable for aggregate quantification directly from different cell culture solutions. Real-process samples and process-sample mimics were used to verify the general aggregate quantification model using two different antibodies. Results showed good prediction ability down to 1% aggregate content. Together with recently published results using MIR for host cell protein and target protein quantification, the results presented here indicate that MIR could provide multi-parameter process information from a single, fast, cost-effective and straightforward measurement. In conclusion, our study demonstrates that MIR is suitable for aggregate quantification in therapeutic antibody purification processes.

Matrix effects during monitoring of antibody and host cell proteins using attenuated total reflection spectroscopy.

Production of recombinant proteins, e.g. antibodies, requires constant real-time monitoring to optimize yield and quality attributes and to respond to changing production conditions, such as host cell protein (HCP) titers. To date, this monitoring of mammalian cell culture-based processes is done using laborious and time consuming enzyme-linked immunosorbent assays (ELISA), two-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis, and chromatography-based systems. Measurements are usually performed off-line, requiring regular sample withdrawal associated with increased contamination risk. As information is obtained retrospectively, the reaction time to adapt to process changes is too long, leading to lower yield and higher costs. To address the resulting demand for continuous online-monitoring systems, we present a feasibility study using attenuated total reflection spectroscopy (ATR) to monitor mAb and HCP levels of NS0 cell culture in situ, taking matrix effects into account. Fifty-six NS0 cell culture samples were treated with polyelectrolytes for semi-selective protein precipitation. Additionally, part of the samples was subjected to filtration prior to analysis, to change the background matrix and evaluate effects on chemometric quantification models. General models to quantify HCP and mAb in both filtered and unfiltered matrix showed lower prediction accuracy compared to models designed for a specific matrix. HCP quantification in the range of 2,000-55,000 ng mL(-1) using specific models was accurate for most samples, with results within the accepted limit of an ELISA assay. In contrast, mAb prediction was less accurate, predicting mAb in the range of 0.2-1.7 g L(-1) . As some samples deviated substantially from reference values, further investigations elucidating the suitability of ATR for monitoring are required.

Host cell protein quantification by Fourier transform mid infrared spectroscopy (FT-MIR).

Process development in up- and downstream processing requires enhanced, non-time-consuming, and non-expensive monitoring techniques to track product purity, for example, the level of endotoxins, viral particles, and host cell proteins (HCPs). Currently, HCP amounts are measured by laborious and expensive HCP-enzyme-linked immunosorbent assay (ELISA) assays best suited for measuring HCP amounts in the low concentration regime. The measurement of higher HCP amounts using this method requires dilution steps, adding dilution errors to the measurement. In this work we evaluated the suitability of attenuated total reflection spectroscopy for HCP quantification in process development, using clarified cell culture fluid from monoclonal antibody producing Chinese hamster ovary-cells after treatment with different polyelectrolytes for semi-selective clarification. Forty undiluted samples were chosen for multivariate data analysis in the middle infrared range and predicted HCP-values were in good agreement with results obtained by an ELISA-assay, suggesting the suitability of this new method for HCP-quantification. As this method is able to quantify HCP titers ranging from approximately at least 20,000-200,000 ng mL(-1), it is suitable especially for monitoring of process development steps with higher HCP concentrations, omitting dilution errors associated with ELISA assays.

Particle packed columns and monolithic columns in high-performance liquid chromatography-comparison and critical appraisal.

The review highlights the fundamentals and the most prominent achievements in the field of high-performance liquid chromatography (HPLC) column development over a period of nearly 50 years. After a short introduction on the structure and function of HPLC columns, the first part treats the major steps and processes in the manufacture of a particle packed column: synthesis and control of particle morphology, sizing and size analysis, packing procedures and performance characterization. The next section is devoted to three subjects, which reflect the recent development and the main future directions of packed columns: minimum particle size of packing, totally porous vs. core/shell particles and column miniaturization. In the last section an analysis is given on an alternative to packed columns-monolithic columns, which have gained considerable attraction. The challenges are: improved packing design based on modeling and simulation for targeted applications, and enhanced robustness and reproducibility of monolithic columns. In the field of miniaturization, particularly in chip-based nano-LC systems, monoliths offer a great potential for the separation of complex mixtures e.g. in life science.

Pore structural characteristics, size exclusion properties and column performance of two mesoporous amorphous silicas and their pseudomorphically transformed MCM-41 type derivatives.

Highly ordered mesoporous silicas such as, mobile composition of matter, MCM-41, MCM-48, and the SBA-types of materials have helped to a large extent to understand the formation mechanisms of the pore structure of adsorbents and to improve the methods of pore structural characterization. It still remains an open question whether the high order, the regularity of the pore system, and the narrow pore size distribution of the materials will lead to a substantial benefit when these materials are employed in liquid phase separation processes. MCM-41 type 10 microm beads are synthesized following the route of pseudomorphic transformation of highly porous amorphous silicas. Highly porous silicas and the pseudomorphically transformed derivatives are characterized by nitrogen sorption at 77 K and by inverse size-exclusion chromatography (ISEC) employing polystyrene standards. Applying the network model developed by Grimes, we calculated the pore connectivity n(T) of the materials. The value of n(T) varies between the percolation threshold of the lattice and values of n(T) > 10, the latter being the limiting value above which the material can be considered to be almost infinitely connected such that the ISEC behavior of the material calculated with the pore network model is the same when calculated with a parallel pore model which assumes an infinite connectivity. One should expect that the pore connectivity is reflected in the column performance, when these native and unmodified materials are packed into columns and tested with low molecular weight analytes in the Normal Phase LC mode. As found in a previous study on monolithic silicas and highly porous silicas, the slope of the plate height (HETP) - linear velocity (u) curve decreased significantly with enhanced pore connectivity of the materials. First results on the pseudomorphically transformed MCM-41 type silicas and their highly porous amorphous precursors showed that (i) the transformation did not change the pore connectivity (within the limits detectable by ISEC) from the starting material to the final product and (ii) the slope of the HETP versus u curve for dibutylphtalate did not change significantly after the pseudomorphic transformation.

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins.

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18

Pore structural characterization of monolithic silica columns by inverse size-exclusion chromatography.

In this work, a parallel pore model (PPM) and a pore network model (PNM) are developed to provide a state-of-art method for the calculation of several characteristic pore structural parameters from inverse size-exclusion chromatography (ISEC) experiments. The proposed PPM and PNM could be applicable to both monoliths and columns packed with porous particles. The PPM and PNM proposed in this work are able to predict the existence of the second inflection point in the experimental exclusion curve that has been observed for monolithic materials by accounting for volume partitioning of the polymer standards in the macropores of the column. The appearance and prominence of the second inflection point in the exclusion curve is determined to depend strongly on the void fraction of the macropores (flow-through pores), (b) the nominal diameter of the macropores, and (c) the radius of gyration of the largest polymer standard employed in the determination of the experimental ISEC exclusion curve. The conditions that dictate the appearance and prominence of the second inflection point in the exclusion curve are presented. The proposed models are applied to experimentally measured ISEC exclusion curves of six silica monoliths having different macropore and mesopore diameters. The PPM and PNM proposed in this work are able to determine the void fractions of the macropores and silica skeleton, the pore connectivity of the mesopores, as well as the pore number distribution (PND) and pore volume distribution (PVD) of the mesopores. The results indicate that the mesoporous structure of all materials studied is well connected as evidenced by the similarities between the PVDs calculated with the PPM and the PNM, and by the high pore connectivity values obtained from the PNM. Due to the fact that the proposed models can predict the existence of the second inflection point in the exclusion curves, the proposed models could be more applicable than other models for ISEC characterization of chromatographic columns with small diameter macropores (interstitial pores) and/or large macropore (interstitial pore) void fractions. It should be noted that the PNM can always be applied without the use of the PPM, since the PPM is an idealization that considers an infinitely connected porous medium and for materials having a low (<6) pore connectivity the PPM would force the PVD to a lower average diameter and larger distribution width as opposed to properly accounting for the network effects present in the real porous medium.