A high-throughput approach to developing and optimizing mixed-mode membrane chromatography for protein purification.

Membrane chromatography has been established as a viable alternative to packed-bed column chromatography for the purification of therapeutic proteins. Purification via membrane chromatography offers key advantages, including higher productivity and reduced buffer usage. Unlike column chromatography purification, the utilization of high-throughput screening in order to reduce development times and material requirements has been a challenge for membrane chromatography. This research focused on the development of a new, high-throughput screening technique for use in screening membrane chromatography conditions for monoclonal antibody purification. The developed screen utilizes a 96-well plate format, thereby allowing for the screening of multiple different membrane conditions at once. For this study, four mixed-mode cation exchange membranes and one cation exchange membrane were evaluated on the plate. The screen is performed in a similar manner to that of a resin slurry plate screen, however, instead of a single loading step, the antibody feed was loaded in 50 mg/ml increments up to a maximum loading of 450 mg/ml. Performing a similar, incremental loading on a resin plate would be impractical, as mixing times are substantially longer due to pore diffusion limitations. However, due to the significantly faster rate of mass transfer for membranes relative to resin, mixing times could be reduced by up to a factor of sixty on the membrane plate. Additional optimization showed that higher hydrophobicity can potentially lead to slower kinetics and mixing times that may need to be adjusted accordingly. The end result is a screen that has been proven to provide results comparable to those obtained on larger-scale membrane purification runs while also enabling exploration of a much greater operating space and significantly reducing the feed materials required.

Streamlined process development procedure incorporating the selection of various stationary phase types established in a mAb aggregate reduction study with different mixed mode ligands.

In biopharmaceutical process development time, cost and reliability are the relevant keywords. During the development of chromatographic processes these targets are challenged by many possible scaffolds, ligands and process parameters. The common response to this diversity is the establishment of platform processes in the development of chromatographic unit operations. However, while developing a platform library to simplify and accelerate chromatographic processes, the potential combination of scaffold, ligands and process parameters need to be characterized. This challenge is addressed in a case study on novel mixed mode (MM) adsorber for the removal of monoclonal antibody (mAb) aggregates. We propose a rigorous strategy to reduce the various experimental design space resulting from possible combinations in scaffolds, backbones and ligands. This strategy is based on theoretical considerations, identification of adsorber selectivity and capacity for the identification of a suitable membrane system. For this system, each potential MM membrane adsorber candidate is investigated in its high molecular weight species reduction potential for a given mAb feed stream and referenced to the performance of Capto™ Adhere. The introduced strategy can reduce the developmental effort in an early stage from three to two possible stationary phases. Thereafter, initial examinations at different ionic capacities enlighten one favorable stationary phase. Finalizing the development strategy procedure by studying five different MM ligands by HTS and confirming the study with a 2-3 MV higher dynamic breakthrough capacity in benchtop experiments and provides an insight in the benefits of a living process platform library.

Process development exploiting competitive adsorption-based displacement effects in monoclonal antibody aggregate removal-A new high-throughput screening procedure for membrane chromatography.

High-throughput screening (HTS) approaches are commonly used to accelerate downstream process development. Although most HTS approaches use batch isothermal data (KP screen) or bind and elute mode as screening procedure, different or new process designs are rarely investigated. In this paper, a mechanistic model case study for the separation of two different two-component solutions was conducted and confirmed prior evidence. With these outcomes, a novel HTS screening procedure was developed including the determination of competitive adsorption-based displacement effects and key parameter identification. The screening procedure employing an overload bind and elute (OBE) mode is presented in a case study dealing with IgG aggregate removal in a typical monoclonal antibody purification step, applying a Sartobind® S membrane adsorber (MA). Based on a MA scale down device, the OBE mode allows the determination of classical process parameters and dynamic effects, such as displacement effects. Competitive adsorption-based displacement effects are visualized by introducing a displacement identifier leading to a displacement process map. Based on this map, the approach is transferred to and confirmed by the OBE recycle experiments with 4.6 and 8.2 ml benchtop scsale devices resulting in 45% reduced IgG monomer and 88% increased higher molecular weight species binding capacities.

Soft synthetic microgels as mimics of mycoplasma.

Artificial model colloids are of special interest in the development of advanced sterile filters, as they are able to efficiently separate pleomorphic, highly deformable and infectious bacteria such as mycoplasma, which, until now, has been considered rather challenging and laborious. This study presents a full range of different soft to super soft synthetic polymeric microgels, including two types with similar hydrodynamic mean diameter, i.e., 180 nm, and zeta potential, i.e., -25 ± 10 mV, but different deformability, synthesized by inverse miniemulsion terpolymerization of acrylamide, sodium acrylate and N,N'-methylenebisacrylamide. These microgels were characterized by means of dynamic, electrophoretic and static light scattering techniques. In addition, the deformability of the colloids was investigated by filter cake compressibility studies during ultrafiltration in dead-end mode, analogously to a study of real mycoplasma, i.e., Acholeplasma laidlawii, to allow for a direct comparison. The results indicate that the variation of the synthesis parameters, i.e., crosslinker content, polymeric solid content and content of sodium acrylate, has a significant impact on the swelling behavior of the microgels in aqueous solution as well as on their deformability under filtration conditions. A higher density of chemical crosslinking points results in less swollen and more rigid microgels. Furthermore, these parameters determine electrokinetic properties of the more or less permeable colloids. Overall, it is shown that these soft synthetic microgels can be obtained with tailor-made properties, covering the size of smallest species of and otherwise similar to real mycoplasma. This is a relevant first step towards the future use of synthetic microgels as mimics for mycoplasma.

Modeling hindered diffusion of antibodies in agarose beads considering pore size reduction due to adsorption.

The aim of this study is to model, describe and predict the mass transfer of IgG as a function of the agarose concentration in the protein A stationary phase, taking into account the influence of adsorption on the pore size. Therefore, particle size distribution, bed and bead porosities were examined by light microscopy, pressure-flow behavior and iSEC. Three agarose protein A stationary phases (2 wt%, 4 wt%, 6 wt%) were investigated. The pore size decreased from 116 nm for 2 wt% to 54 nm for 6 wt% and the porosity for the target molecule IgG was reduced by 25%. A shrinking core model approach was used to assess the influence of IgG adsorption on the pore size of the stationary phase and the diffusivity of IgG. Due to IgG adsorption, the pore diameter reduced by 24 nm, which is approximately two times its hydrodynamic diameter. Effective pore diffusivities of IgG were obtained by fitting the general rate model to breakthrough curves. They were in the range between 3.96·10-12m2/s and 6.5·10-12m2/s, decreasing as the agarose concentration increased. The DBC1% has a maximum for the 4 wt% agarose gel, showing optimal tradeoffs between accessibility, specific surface and diffusive mass transfer for IgG. A simple geometrical model was developed to describe the change in pore and filament diameters due to adsorption. The diffusion measured in protein A agarose beads can be described by a modification of the Ogston model. This enables the diffusion measured in protein A agarose networks to be predicted.

High throughput screening setup of a scale-down device for membrane chromatography-aggregate removal of monoclonal antibodies.

In biopharmaceutical process development, resin-based high throughput screening (HTS) is well known for overcoming experimental limitations by permitting automated parallel processing at miniaturized scale, which results in fast data generation and reduced feed consumption. For membrane adsorber (MA), HTS solutions have so far only been available to a partial extent. Three case studies were performed with the aim of aligning HTS applications for MAs with those established for column chromatography: Process parameter range determination, mechanistic modeling (MM), and scalability. In order to exploit the MA typically features, such as high mass transfer and easy scalability, for scalable high throughput process development, a scale-down device (SDD) for MA was developed. Its applicability is confirmed for a monoclonal antibody aggregate removal step. The first case study explores the experimental application of the SDD developed. It uses bind and elute mode and variations of pH and salt concentration to obtain process operation windows for ion-exchange MAs Sartobind® S and Q. In the second case study, we successfully developed a mechanistic model based on parameters obtained from the SDD-HTS setup. The results proved to validate the use of the SDD developed for parameter estimation and thus model-based process development. The third case study shows the transferability and scalability of data from the SDD-HTS setup using both a direct scale factor and MM. Both approaches show good applicability with a deviation below 20% in the prediction of 10% dynamic breakthrough capacity and reliable scale-up from 0.42 to 800 ml.

Investigation of microbial cell deformability by filter cake compressibility using ultrafiltration membranes.

This study presents the investigation of deformability of various microbial cells in terms of filter cake compressibility during cake filtration using ultrafiltration membranes in dead-end mode. The examined microbial cells include mycoplasma, Gram-positive and Gram-negative bacteria, and Pseudomonas aeruginosa phage PP7. Polystyrene particles were used as an incompressible reference. The compressibility results were correlated to the deformability of a microbial cell, induced by its cell envelope. To determine the deformability of the different microbial cells under different process conditions, their cake resistance was measured under varying pressures from 10 to 250 kPa and temperatures from 2 to 35 °C. In addition, the influence of different culture media on the cell properties of Acholeplasma laidlawii and its behavior under different pressure and temperature was determined. The results of the pressure and temperature experiments revealed that Gram-positive S. epidermidis was found to be relatively stiff due to the thickness of the peptidoglycan layer, under different pressure and temperature conditions. No significant increase of the specific cake resistance of S. epidermidis could be determined. B. diminuta however showed a high deformation tendency when the pressure was increased indicating relatively soft cells. Mycoplasma A. laidlawii cells cultivated in three different media showed a different, but significant, effect of pressure and temperature.

Investigation of fouling mechanisms of virus filters during the filtration of protein solutions using a high throughput filtration screening device.

The downstream process development of novel antibodies (Abs) is often challenged by virus filter fouling making a better understanding of the underlying mechanisms highly desirable. The present study combines the protein characterization of different feedstreams with their virus filtration performance using a novel high throughput filtration screening system. Filtration experiments with Ab concentrations of up to 20 g/L using either low interacting or hydrophobically interacting pre-filters indicate the existence of two different fouling mechanisms, an irreversible and a reversible one. At the molecular level, size exclusion chromatography revealed that the presence of large amount of high molecular weight species-considered as irreversible aggregates-correlates with irreversible fouling that caused reduced Ab throughput. Results using dynamic light scattering show that a concentration dependent increase of the mean hydrodynamic diameter to the range of dimers (17 nm at 20 g/L) together with a negative DLS interaction parameter kD (-18 mL/g) correlate with the propensity to form reversible aggregates and to cause reversible fouling, probably by a decelerated Ab transport velocity within the virus filter. The two fouling mechanisms are further supported by buffer flush experiments. Finally, concepts for reversible and irreversible fouling mechanisms are discussed together with strategies for respective fouling mitigation. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2776, 2019.

Investigation of virus retention by size exclusion membranes under different flow regimes.

Virus removal by filter membranes is regarded as a robust and efficient unit operation, which is frequently applied in the downstream processing of biopharmaceuticals. The retention of viruses by virus filtration membranes is predominantly based on size exclusion. However, recent results using model membranes and bacteriophage PP7 point to the fact that virus retention can also significantly be influenced by adsorptive interactions between virus, product molecules, and membranes. Furthermore, the impact of flow rate and flow interruptions on virus retention have been studied and responsible mechanisms discussed. The aim of this investigation was to gain a holistic understanding of the underlying mechanisms for virus retention in size exclusion membranes as a function of membrane structure and membrane surface properties, as well as flow and solution conditions. The results of this study contribute to the differentiation between size exclusion and adsorptive effects during virus filtration and broaden the current understanding of mechanisms related to virus breakthroughs after temporary flow interruptions. Within the frame of a Design of Experiments approach it was found that the level of retention of virus filtration membranes was mostly influenced by the membrane structure during typical process-related flow conditions. The retention performance after a flow interruption was also significantly influenced by membrane surface properties and solution conditions. While size exclusion was confirmed as main retention mechanism, the analysis of all results suggests that especially after a flow interruption virus retention can be influenced by adsorptive effects between the virus and the membrane surface. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2747, 2019.

Retention of Acholeplasma laidlawii by Sterile Filtration Membranes: Effect of Cultivation Medium and Filtration Temperature.

This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma Acholeplasma laidlawii cultivated in five different cultivation media. The influence of relevant filtration process parameters, in particular transmembrane pressure and filtration temperature, on their respective retention was tested. The impact of the filtration temperature was further evaluated for the Gram-negative bacteria species Brevundimonas diminuta, the Gram-positive bacteria species Staphylococcus epidermidis, the Pseudomonas phage PP7, and the mycoplasma species Mycoplasma orale The findings were correlated to the different mechanical properties of the particles, especially also with respect to the different bacterial cell envelopes found in those species. This study suggests that mycoplasma, surrounded by a flexible lipid bilayer, are significantly susceptible to changes in temperature, altering the stiffness of the cell envelope. Mycoplasma retention could thus be increased significantly by a decreased filtration temperature. In contrast, Gram-negative and Gram-positive bacteria species, with a cell wall containing a cross-linked peptidoglycan layer, as well as bacteriophages PP7 exhibiting a rigid protein capsid, did not show a temperature-dependent retention within the applied filtration temperatures between 2 and 35 °C. The trends of the retention of A. laidlawii with increasing temperature and transmembrane pressure were independent of cultivation media. Data obtained with mycoplasma M. orale suggest that the trend of mycoplasma retention at different filtration temperatures is also independent of the membrane pore size and thus retention level.LAY ABSTRACT: Media in biopharmaceutical processes are sterile-filtered to prevent them from bacterial contamination. Mycoplasma represent a relevant class of bacteria. In this publication it is shown that mycoplasma cell size depends on the media they are cultivated in. Membranes used for sterile filtration retain bacteria predominantly by size exclusion. Thus, an altered cell size can result in different retention values. Another characteristic of mycoplasma is the flexible lipid bilayer and the absence of a rigid cell wall. The lipid bilayer can undergo a phase transition from a gel to a liquid-crystal phase at a certain temperature, which makes it stiffer at lower temperatures. A higher stiffness can result in higher retention values during filtration, as the deformability of the mycoplasma cell is lower and the cell does not squeeze through the membrane pores. ABBREVIATIONS: ALCM: A. laidlawii culture medium; ASTM: American Society for Testing and Materials; ATCC: American Type Culture Collection; CFU/mL: colony-forming units per milliliter; DLS: Dynamic light scattering; LRV: Log reduction value; PES: Polyethersulfone; PFU/mL: Plaque-forming units per milliliter; PSD: Particle size distribution; PVP: Polyvinylpyrrolidone; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SLB: Saline lactose broth; TMP: Transmembrane pressure; TSB: Tryptic soy broth.