Purification Of Biopharmaceuticals Research Articles

Chromatography media and purification processes for complex and super-large biomolecules: A review.

The biopharmaceutical industry has been one of the most dynamic industries in the world. New biopharmaceuticals are constantly developed, especially for complex and super-large biomolecules including antibodies, virus-like particles (VLPs), viral vectors, DNA, mRNA, and are very promising in drugs, vaccines, cell and gene therapy. Due to complex and unstable structures, as well as low concentration, it is very difficult to purify these complex and super-large biomolecules. Chromatography is the most widely used purification technique in bioseparation, and chromatography media is the key material. This review gives a comprehensive analysis of chromatography media and purification processes for complex and super-large biomolecules. A detailed summary of tailor-made chromatography media is first provided, including particle size and its uniformity, pore structure, spacer arm and polymer grafting, and new ligands and special separation mechanisms. Then the current choices and trends of purification processes for vaccines, VLPs, DNA, mRNA, antibodies and modified therapeutic proteins are reviewed. Finally, a brief overview of continuous biochromatography and computer-assisted chromatographic method development is provided. We hope this review will provide some useful guidance for design of chromatography media and purification of complex biopharmaceuticals.

Proteomics-based method to comprehensively model the removal of host cell protein impurities.

Mechanistic models mostly focus on the target protein and some selected process- or product-related impurities. For a better process understanding, however, it is advantageous to describe also reoccurring host cell protein impurities. Within the purification of biopharmaceuticals, the binding of host cell proteins to a chromatographic resin is far from being described comprehensively. For a broader coverage of the binding characteristics, large-scale proteomic data and systems level knowledge on protein interactions are key. However, a method for determining binding parameters of the entire host cell proteome to selected chromatography resins is still lacking. In this work, we have developed a method to determine binding parameters of all detected individual host cell proteins in an Escherichia coli harvest sample from large-scale proteomics experiments. The developed method was demonstrated to model abundant and problematic proteins, which are crucial impurities to be removed. For these 15 proteins covering varying concentration ranges, the model predicts the independently measured retention time during the validation gradient well. Finally, we optimized the anion exchange chromatography capture step in silico using the determined isotherm parameters of the persistent host cell protein contaminants. From these results, strategies can be developed to separate abundant and problematic impurities from the target antigen.

In-line fiber optical sensor for detection of IgG aggregates in affinity chromatography

Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.

Competitive protein adsorption in isocratic anion exchange chromatography

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.

Investigating and Optimizing Insulin Partitioning with Conjugated Au Nanoparticles in Aqueous Two-Phase Systems Using Response Surface Methodology.

This study investigated the impact of bioconjugation on the partitioning of insulin, a clinically valuable protein, in an aqueous two-phase system. Gold nanoparticles of different sizes were synthesized and conjugated with insulin. Analysis of the conjugated insulin showed that the insulin remains fully active. Conjugated gold nanoparticles (AuNPs/insulin) were used in polyethylene glycol (PEG)-dextran aqueous two-phase systems to investigate the effect of pH, PEG and dextran molecular weights, PEG and dextran concentrations, AuNPs/insulin dosage, and nanoparticle size on the partition coefficient. These systems were chosen for their biocompatibility and low toxicity. Response surface methodology with D-optimal design was used to model and optimize these systems and their affected parameters. At the optimum condition of a pH = 8 system containing 21% PEG 4000, 5% dextran 100,000, and 100 IU AuNPs/insulin, the partition coefficient of AuNPs/insulin was found to be 192.96, which is in agreement with the empirical partition coefficient of 189.2. This is significantly higher than the partition coefficient of free insulin in a similar system. This approach could be used to overcome limitations in the feasibility of aqueous two-phase systems for industrial-scale purification of biomolecules and biopharmaceuticals.

Toward microfluidic continuous-flow and intelligent downstream processing of biopharmaceuticals.

Biopharmaceuticals have emerged as powerful therapeutic agents, revolutionizing the treatment landscape for various diseases, including cancer, infectious diseases, autoimmune and genetic disorders. These biotherapeutics pave the way for precision medicine with their unique and targeted capabilities. The production of high-quality biologics entails intricate manufacturing processes, including cell culture, fermentation, purification, and formulation, necessitating specialized facilities and expertise. These complex processes are subject to rigorous regulatory oversight to evaluate the safety, efficacy, and quality of biotherapeutics prior to clinical approval. Consequently, these drugs undergo extensive purification unit operations to achieve high purity by effectively removing impurities and contaminants. The field of personalized precision medicine necessitates the development of novel and highly efficient technologies. Microfluidic technology addresses unmet needs by enabling precise and compact separation, allowing rapid, integrated and continuous purification modules. Moreover, the integration of intelligent biomanufacturing systems with miniaturized devices presents an opportunity to significantly enhance the robustness of complex downstream processing of biopharmaceuticals, with the benefits of automation and advanced control. This allows seamless data exchange, real-time monitoring, and synchronization of purification steps, leading to improved process efficiency, data management, and decision-making. Integrating autonomous systems into biopharmaceutical purification ensures adherence to regulatory standards, such as good manufacturing practice (GMP), positioning the industry to effectively address emerging market demands for personalized precision nano-medicines. This perspective review will emphasize on the significance, challenges, and prospects associated with the adoption of continuous, integrated, and intelligent methodologies in small-scale downstream processing for various types of biologics. By utilizing microfluidic technology and intelligent systems, purification processes can be enhanced for increased efficiency, cost-effectiveness, and regulatory compliance, shaping the future of biopharmaceutical production and enabling the development of personalized and targeted therapies.

Automated calibration and in-line measurement of product quality during therapeutic monoclonal antibody purification using Raman spectroscopy.

Current manufacturing and development processes for therapeutic monoclonal antibodies demand increasing volumes of analytical testing for both real-time process controls and high-throughput process development. The feasibility of using Raman spectroscopy as an in-line product quality measuring tool has been recently demonstrated and promises to relieve this analytical bottleneck. Here, we resolve time-consuming calibration process that requires fractionation and preparative experiments covering variations of product quality attributes (PQAs) by engineering an automation system capable of collecting Raman spectra on the order of hundreds of calibration points from two to three stock seed solutions differing in protein concentration and aggregate level using controlled mixing. We used this automated system to calibrate multi-PQA models that accurately measured product concentration and aggregation every 9.3 s using an in-line flow-cell. We demonstrate the application of a nonlinear calibration model for monitoring product quality in real-time during a biopharmaceutical purification process intended for clinical and commercial manufacturing. These results demonstrate potential feasibility to implement quality monitoring during GGMP manufacturing as well as to increase chemistry, manufacturing, and controls understanding during process development, ultimately leading to more robust and controlled manufacturing processes.

L-Arginine sulfate reduces irreversible protein binding in immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that ‘soft’ proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.

Characterisation of the E. coli HMS174 and BLR host cell proteome to guide purification process development.

Mass-spectrometry-based proteomics is increasingly employed to monitor purification processes or to detect critical host cell proteins in the final drug substance. This approach is inherently unbiased and can be used to identify individual host cell proteins without prior knowledge. In process development for the purification of new biopharmaceuticals, such as protein subunit vaccines, a broader knowledge of the host cell proteome could promote a more rational process design. Proteomics can establish qualitative and quantitative information on the complete host cell proteome before purification (i.e., protein abundances and physicochemical properties). Such information allows for a more rational design of the purification strategy and accelerates purification process development. In this study, we present an extensive proteomic characterisation of two E.coli host cell strains widely employed in academia and industry to produce therapeutic proteins, BLR and HMS174. The established database contains the observed abundance of each identified protein, information relating to their hydrophobicity, the isoelectric point, molecular weight, and toxicity. These physicochemical properties were plotted on proteome property maps to showcase the selection of suitable purification strategies. Furthermore, sequence alignment allowed integration of subunit information and occurrences of post-translational modifications from the well-studied E.coli K12 strain.

Next generation multimodal chromatography resins via an iterative mapping approach: Chemical diversity, high-throughput screening, and chromatographic modelling

Multimodal chromatography resins are becoming a key tool in the purification of biomolecules. The main objective of this research was the establishment of an iterative framework for the rapid development of new multimodal resins to provide novel selectivity for the future purification challenges. A large chemically diverse virtual library of 100 multimodal Capto™ MMC ligand analogues was created, and a broad array of chemical descriptors were calculated for each ligand in silico. Principal component analysis (PCA) was used to map the chemical diversity and guide selection of ligands for synthesis and coupling to the Capto ImpRes agarose base matrix. Twelve new ligands were prepared in two groups: ‘group one’ consist of L00-L07 and ‘group two’ consist of L08-L12. These ligands are diverse in the influence of varied secondary interactions such as hydrophobic interactions, H-bonding, etc. Additional resin prototypes were also prepared to look at the chromatographic impact of ligand density variation. High-throughput plate-based studies were performed for parallel resin screening for batch-binding of six model proteins at different chromatographic binding pH and sodium chloride concentration conditions. Principal component analysis of the binding data provided a chromatographic diversity map leading to the identification of ligands with improved binding. Further, the new ligands have improved separation resolution between a monoclonal antibody (mAb1) and product related impurities, a Fab fragment and high molecular weight (HMW) aggregates, using linear salt gradient elutions. To quantify the importance of secondary interactions, analysis of the retention factor of mAb1 on the ligands at various isocratic conditions lead to estimations of (a) the total number of water molecules and counter salt ions released during adsorption, and (b) hydrophobic contact area (HCA). The iterative mapping approach of chemical and chromatography diversity maps described in the paper proves to be a promising method for identifying new chromatography ligands for biopharmaceutical purification challenges.

An automated buffer management system for small-scale continuous downstream bioprocessing

Buffer management for biopharmaceutical purification processes include buffer preparation, storage of buffers and restocking the buffers when needed. This is usually performed manually by the operators for small scale operations. However, buffer management can become a bottleneck when running integrated continuous purification processes for prolonged times, even at small scale. To address this issue, a buffer management system for the application in continuous lab-scale bioprocessing is presented in this paper. For this purpose, an ÄKTA™ explorer chromatography system was reconfigured to perform the buffer formulation. The system formulated all buffers from stock solutions and water according to pre-specified recipes. A digital twin of the physical system was introduced in the research software Orbit, written in python. Orbit was also used for full automation and control of the buffer system, which could run independently without operator input and handle buffer management for one or several connected buffer-consuming purification systems. The developed buffer management system performed automatic monitoring of buffer volumes, buffer order handling as well as buffer preparation and delivery. To demonstrate the capability of the developed system, it was integrated with a continuous downstream process and supplied all 9 required buffers to the process equipment during a 10-day operation. The buffer management system processed 55 orders and delivered 38 L of buffers, corresponding to 20% of its capacity. The pH and conductivity profiles observed during the purification steps were consistent across the cycles. The deviation in conductivity and pH from the measured average value was within ±0.89% in conductivity and ±0.045 in pH, well within the typical specification for buffer release, indicating that the prepared buffers had the correct composition. The operation of the developed buffer management system was robust and fully automated, and provides one solution to the buffer management bottleneck on lab scale for integrated continuous downstream bioprocessing.

Standardized method for mechanistic modeling of multimodal anion exchange chromatography in flow through operation

Multimodal chromatography offers an increased selectivity compared to unimodal chromatographic methods and is often employed for challenging separation tasks in industrial downstream processing (DSP). Unfortunately, the implementation of multimodal polishing into a generic downstream platform can be hampered by non-robust platform conditions leading to a time and cost intensive process development. Mechanistic modeling can assist experimental process development but readily applicable and easy to calibrate multimodal chromatography models are lacking. In this work, we present a mechanistic modeling aided approach that paves the way for an accelerated development of anionic mixed-mode chromatography (MMC) for biopharmaceutical purification. A modified multimodal isotherm model was calibrated using only three chromatographic experiments and was employed in the retention prediction of four antibody formats including a Fab, a bispecific, as well as an IgG1 and IgG4 antibody subtype at pH 5.0 and 6.0. The chromatographic experiments were conducted using the anionic mixed-mode resin Capto adhere at industrial relevant process conditions to enable flow through purification. An existing multimodal isotherm model was reduced to hydrophobic interactions in the linear range of the adsorption isotherm and successfully employed in the simulation of six chromatographic experiments per molecule in concert with the transport dispersive model (TDM). The model reduction to only three parameters did prevent structural parameter non-identifiability and enabled an analytical isotherm parameter determination that was further refined by incorporation of size exclusion effects of the selected multimodal resin. During the model calibration, three linear salt gradient elution experiments were performed for each molecule followed by an isotherm parameter uncertainty assessment. Lastly, each model was validated with a set of step and isocratic elution experiments. This standardized modeling approach facilitates the implementation of multimodal chromatography as a key unit operation for the biopharmaceutical downstream platform, while increasing the mechanistic insight to the multimodal adsorption behavior of complex biologics.

Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

An Experimental and Modeling Combined Approach in Preparative Hydrophobic Interaction Chromatography

Chromatography is a technique widely used in the purification of biopharmaceuticals, and generally consists of several chromatographic steps. In this work, Hydrophobic Interaction Chromatography (HIC) is investigated as a polishing step for the purification of therapeutic proteins. Adsorption mechanisms in hydrophobic interaction chromatography are still not completely clear and a limited amount of published data is available. In addition to new data on adsorption isotherms for some proteins (obtained both by high-throughput and frontal analysis method), and a comparison of different models proposed in the literature, two different approaches are compared in this work to investigate HIC. The predictive approach exploits an in-house code that simulates the behavior of the component in the column using the model parameters found from the fitting of experimental data. The estimation approach, on the other hand, exploits commercial software in which the model parameters are found by the fitting of a few experimental chromatograms. The two approaches are validated on some bind-elute runs: the predictive approach is very informative, but the experimental effort needed is high; the estimation approach is more effective, but the knowledge gained is lower. The second approach is also applied to an in-development industrial purification process and successfully resulted in predicting the behavior of the system, allowing for optimization with a reduction in the time and amount of sample needed.

Binary separation control in preparative gradient chromatography using iterative learning control

Purification of biopharmaceuticals has shifted toward continuous and integrated processes, in turn bringing along a need for monitoring and control to maintain a desired separation between the target pharmaceutical and any impurities it may carry. In this study, a cycle-to-cycle control of the retention volumes of two compounds in a chromatographic, ion exchange purification step was developed, allowing the process to maintain the desired retention volumes in the separation. The controller made use of a model-based, multivariate iterative learning control (ILC) algorithm that used a quadratic-criterion objective function for optimal set point control, along with feed-forward control based on direct model inversion for preemptive control of set point changes. The model was calibrated using 3 experiments, allowing for fast setup. The controller was tested by introducing three different disturbances to a sequence of otherwise identical ion exchange separation processes: a change in the salt concentration of the elution buffer, a change in set point, and a change in the pH of the elution buffer. It was capable of correcting for all disturbances within at most 3 cycles, proving its efficacy. The successful application of ILC for separation control in biopharmaceutical purification paves the way for the development of further ILC-based control strategies within the field, as well as combination with other control strategies.

Automated ion exchange chromatography screening combined with in silico multifactorial simulation for efficient method development and purification of biopharmaceutical targets.

Bioprocess development of increasingly challenging therapeutics and vaccines requires a commensurate level of analytical innovation to deliver critical assays across functional areas. Chromatography hyphenated to numerous choices of detection has undeniably been the preferred analytical tool in the pharmaceutical industry for decades to analyze and isolate targets (e.g., APIs, intermediates, and byproducts) from multicomponent mixtures. Among many techniques, ion exchange chromatography (IEX) is widely used for the analysis and purification of biopharmaceuticals due to its unique selectivity that delivers distinctive chromatographic profiles compared to other separation modes (e.g., RPLC, HILIC, and SFC) without denaturing protein targets upon isolation process. However, IEX method development is still considered one of the most challenging and laborious approaches due to the many variables involved such as elution mechanism (via salt, pH, or salt-mediated-pH gradients), stationary phase's properties (positively or negatively charged; strong or weak ion exchanger), buffer type and ionic strength as well as pH choices. Herein, we introduce a new framework consisting of a multicolumn IEX screening in conjunction with computer-assisted simulation for efficient method development and purification of biopharmaceuticals. The screening component integrates a total of 12 different columns and 24 mobile phases that are sequentially operated in a straightforward automated fashion for both cation and anion exchange modes (CEX and AEX, respectively). Optimal and robust operating conditions are achieved via computer-assisted simulation using readily available software (ACD Laboratories/LC Simulator), showcasing differences between experimental and simulated retention times of less than 0.5%. In addition, automated fraction collection is also incorporated into this framework, illustrating the practicality and ease of use in the context of separation, analysis, and purification of nucleotides, peptides, and proteins. Finally, we provide examples of the use of this IEX screening as a framework to identify efficient first dimension (1D) conditions that are combined with MS-friendly RPLC conditions in the second dimension (2D) for two-dimensional liquid chromatography experiments enabling purity analysis and identification of pharmaceutical targets.

Ultrahigh-speed, ultrahigh-resolution preparative separation of protein biopharmaceuticals using membrane chromatography.

This paper discusses ultrahigh-speed, ultrahigh-resolution preparative protein separation using an in-house designed membrane chromatography device. The performance of the membrane chromatography device was systematically compared with an equivalent resin-packed preparative column. Experiments carried out using model proteins showed that membrane chromatography gave more than four times greater resolution than the preparative column, while at the same time being more than 19 times faster. Membrane chromatography was therefore a better option, not only in terms of higher productivity but also in terms of higher product purity. Membrane chromatography was also superior in terms of resolving and presenting tracer impurity peaks in the chromatogram. Experiments carried out using monoclonal antibody samples showed that membrane chromatography was suitable for ultrahigh speed, and ultrahigh resolution fractionation of charge variants. This paper highlights and explains the need for proper device design for enabling the use of membrane chromatography for the efficient purification of protein biopharmaceuticals.

Use of Microfluidic Capillary Electrophoresis for the Determination of Multi-Component Protein Adsorption Isotherms: Application to High-Throughput Analysis for Hydrophobic Interaction Chromatography.

Chromatography is a widely used separation process for purification of biopharmaceuticals that is able to obtain high purities and concentrations. The phenomena that occur during separation, mass transfer and adsorption are quite complex. To better understand these phenomena and their mechanisms, multi-component adsorption isotherms must be investigated. High-throughput methodologies are a very powerful tool to determine adsorption isotherms and they waste very small amounts of sample and chemicals, but the quantification of component concentrations is a real bottleneck in multi-component isotherm determination. The behavior of bovine serum albumin, Corynebacterium diphtheriae CRM197 protein and lysozyme, selected as model proteins in binary mixtures with hydrophobic resin, is investigated here. In this work we propose a new method for determining multi-component adsorption isotherms using high-throughput experiments with filter plates, by exploiting microfluidic capillary electrophoresis. The precision and accuracy of the microfluidic capillary electrophoresis platform were evaluated in order to assess the procedure; they were both found to be high and the procedure is thus reliable in determining adsorption isotherms for binary mixtures. Multi-component adsorption isotherms were determined with a totally high-throughput procedure that turned out to be a very fast and powerful tool. The same procedure can be applied to every kind of high-throughput screening.

Plasmid processing for mRNA, gene therapy and other vector applications

<h3>Watch the video or read the poster to learn:</h3> <ul><li>Novel three-step purification process for plasmid (p)DNA </li><li>A new high productivity multimodal resin designed specifically for pDNA purification</li><li>Comparison between legacy and novel purification process in terms of productivity </li><li>How a novel fibro format could offer even greater productivity gains in the future</li><div class="authors authors-lg focus-visible" data-focus-visible-added=""><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Headshopt copy.png" data-image="1"></div><div class="author-det"> <b>Henrik Ihre</b> has his roots in biopharma, leadership and product development for the biopharma downstream industry in specific. He is motivated by bringing new solutions and manufacturing of new pharmaceuticals for patients developed by partners of Cytiva. He has been the Director of Strategic Technologies since March 2020 with specific knowledge and background in the downstream purification of biopharmaceuticals for over 20 years.&nbsp; </div></div></div></ul>

Fast and high-resolution purification of a PEGylated protein using a z2 laterally-fed membrane chromatography device

PEGylated proteins comprise a class of value-added biopharmaceuticals. High-resolution separation techniques are required for the purification of these molecules. In this study, we discuss the application of a newly developed z2 laterally-fed membrane chromatography (or z2LFMC) device for carrying out high-resolution purification of a PEGylated protein drug. The device used in the current study contained a stack of anion exchange (Q) membranes. The membrane bed-height of this z2LFMC device being small, it could be operated at very high flow rates, at relatively low back pressures. The primary goal was to speedily and efficiently separate a mono-PEGylated protein from impurities present in the PEGylation reaction mixture. A resin-based anion exchange column having the same ligand and bed-volume was used as the control device. The purification performance of the z2LFMC device and the control column were compared terms of resolution, recovery and purity. The z2LFMC device outperformed the control column in terms of every metric compared in this study. Higher purity (85.4% as opposed to 77.9%) and higher recovery (28% greater) of the target mono-PEGylated protein were obtained using the z2LFMC device at 20-time higher speed. These results clearly demonstrate that the z2LFMC device could be a faster and more efficient alternative to resin-based columns for purification of biopharmaceuticals.