A tool for increasing the lifetime of chromatography resins

There is a steadily increasing demand for speed, cost efficiency, and process understanding within biopharmaceutical process development. To match this, a high-throughput method for screening of cleaning-in-place (CIP) conditions for chromatography resins has been developed. The methodology includes fouling of MabSelect SuRe chromatography resin in 96-well filter plates, cleaning of the fouled resin by incubation in different CIP agents, and finally, analysis of the residual impurities on the resin after cleaning. This article describes the improvements that transformed the method from low throughput and significant manual interference to a totally automated method with high throughput and good reproducibility.     

A mechanistic study of in situ chemical cleaning-in-place for a PTFE flat sheet membrane: fouling mitigation and membrane characterization

This study aimed at unfolding the role and mechanisms of chemically enhanced cleaning-in-place (CIP) regimes in fouling control of polytetrafluoroethylene (PTFE) made flat sheet (FS) membrane bio-reactors (MBRs). The trans-membrane pressure (TMP) was successfully maintained below 10 kPa using a daily CIP regime consisting of 100 to 600 mg l^?1 of NaOCl and cake layer resistance control was shown to be critical for effective high-flux MBR operation. In contrast, in the control unit without the CIP, the TMP exceeded 35 kPa at a flux of 40 LMH. The extracellular polymeric substances associated with proteins (EPS_protein) were also controlled effectively with a daily application of the CIP to the fouled membrane. Moreover, the CIP prompted a thinner and looser bio-cake layer on the membrane surface, suggesting that in situ CIP can be a favorable method to control FS membrane fouling at high-flux MBR operation.     

High-throughput process development: determination of dynamic binding capacity using microtiter filter plates filled with chromatography resin

Steadily increasing demand for more efficient and more affordable biomolecule-based therapies put a significant burden on biopharma companies to reduce the cost of R&D activities associated with introduction of a new drug to the market. Reducing the time required to develop a purification process would be one option to address the high cost issue. The reduction in time can be accomplished if more efficient methods/tools are available for process development work, including high-throughput techniques. This paper addresses the transitions from traditional column-based process development to a modern high-throughput approach utilizing microtiter filter plates filled with a well-defined volume of chromatography resin. The approach is based on implementing the well-known batch uptake principle into microtiter plate geometry. Two variants of the proposed approach, allowing for either qualitative or quantitative estimation of dynamic binding capacity as a function of residence time, are described. Examples of quantitative estimation of dynamic binding capacities of human polyclonal IgG on MabSelect SuRe and of qualitative estimation of dynamic binding capacity of amyloglucosidase on a prototype of Capto DEAE weak ion exchanger are given. The proposed high-throughput method for determination of dynamic binding capacity significantly reduces time and sample consumption as compared to a traditional method utilizing packed chromatography columns without sacrificing the accuracy of data obtained.     

Rapid quantitation of monoclonal antibody N-glyco-occupancy and afucosylation using mass spectrometry

N-glyco-occupancy and afucoslyation level are two important quality attributes associated with N-glycosylation of therapeutic monoclonal antibodies (mAbs). We report here a fast mass spectrometry-based workflow for quantification of N-glycan site-occupancy and afucoslyation level of mAbs with improved throughput, precision, sensitivity and robustness. This method uses the deglycosylation after the first GlcNAc and inter-chain reduction of the mAbs, followed by liquid chromatography/mass spectrometry (LC-MS) analysis. The entire process can be completed within one hour, which provides a rapid quantitation of N-glyco-occupancy and afucosylation to support high-throughput cell line selection and process development for mAb biopharmaceuticals.     

A label-free methodology for selective protein quantification by means of absorption measurements

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Using a new label-free and non-invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements, we show that the analytical throughput for selective protein quantification can be increased significantly. An analytical assay based on this new methodology was shown to generate very precise results. Further, the assay was successfully applied as analytics for a resin screening performed in HTE mode. The increase in analytical throughput was obtained without decreasing the level of information when compared to analytical chromatography. This proves its potential as a valuable analytical tool in conjugation with high throughput process development (HTPD). Further, fast selective protein quantification can enhance process control in a commercial production environment and, hence, minimize the need for off-line release analysis.     

High throughput chromatography and analytics can inform viral clearance capabilities during downstream process development for biologics

High throughput process development (HTPD) using liquid handling robotics and RoboColumns is an established methodology in downstream process development to screen chromatography resins and optimize process designs to meet target product profiles. However, HTPD is not yet widely available for use in viral clearance capability of the resin due to a variety of constraints. In the present study, a BSL-1-compatible, non-infectious MVM model, MVM-VLP, was tested for viral clearance assessment with various resin and membrane chromatography operations in a HTPD mode. To detect the MVM-VLP in the high throughput experiments, an electrochemiluminescence immunoassay (ECLIA) assay was developed with up to 5 logs of dynamic range. Storage time suitability of MVM-VLP solutions in various buffer matrices, in the presence or absence of a glycoprotein vaccine candidate, were assessed. Then, MVM-VLP and a test article monoclonal antibody (mAb) were used in a HTPD design that included commercially available ion exchange media chemistries, elucidating a wide variety of viral clearance ability at different operating conditions. The methodologies described herein have the potential to be a part of the process design stage in biologics manufacturing process development, which in turn can reduce risk associated with viral clearance validation studies.     

High-throughput screening of chromatographic separations: I. Method development and column modeling

The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 microL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps.     

Recent developments in chromatographic purification of biopharmaceuticals

Over the last several decades, researchers have time and again proposed use of non-chromatographic methods for processing of biotherapeutic products. However, chromatography continues to be the backbone of downstream processing, particularly at process scale. There are many reasons for this, critical ones being the unparalleled scalability, robustness, and selectivity that process chromatography offers over its peers. It is no surprise then that process chromatography has been a topic of major developments in resin matrix, ligand chemistry, modalities, high throughput process development, process modelling, and approaches for control. In this review, we attempt to summarize major developments in the above-mentioned areas. Greater significance has been given to advancements in the last 5 years (2013–2017).     

High-throughput protein purification using an automated set-up for high-yield affinity chromatography

One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.     

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.     

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40°C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60°C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.     

Parallel, high-throughput purification of recombinant antibodies for in vivo cell assays

We describe a method for high-throughput, parallel purification of secreted proteins to analyse large numbers of protein samples in cell-based assays for the discovery of protein therapeutics. The procedure is generic and capable of 96 parallel purifications and compatible, in both yield and purity, with a wide assay range. By optimising expression and purification steps as well as using novel hardware, in particular a chromatography press capable to purify target proteins from viscous media, we exemplify the process for the generation of single-chain Fv antibody fragments (scFv) and the purification of full-length IgG. The described process can operate robustly with a throughput of over 2,000 samples per month.     

Development and application of an automated, low-volume chromatography system for resin and condition screening

A small-volume chromatography system was developed for rapid resin and parameter screening and applied to the purification of a therapeutic monoclonal antibody from a key product-related impurity. Accounting for constraints in peripheral volume, gradient formation, column integrity, and fraction collection in microtiter plates, the resulting system employed 2-mL columns and was successfully integrated with plate-based methods for rapid sample analysis (e. g., use of automated liquid handlers, plate readers, and HPLC). Several cation-exchange chromatography resins were screened using automated programs and tailored gradients for the combination of a particular resin and a given antibody feedstock produced during Phase 1 development. Results from the tailored gradient runs were used to select a resin, and to arrive at efficient stepwise elution schedules for the chosen resin. By maintaining a constant residence time, final operating parameters were successfully scaled to representative bed heights and column diameters up to 2.6 cm (106 mL). This approach significantly improved throughput while reducing development time and material consumption.     

Stability towards alkaline conditions can be engineered into a protein ligand

One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting in replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.     

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40 °C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60 °C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.     

Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography

A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.     

High-throughput screening of chromatographic separations: IV. Ion-exchange

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.     

SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications

Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry.     

Development and validation of high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of loratadine and desloratadine in human plasma

As a continuation of effort to improve our high flow on-line bioanalytical approach for high-throughput quantification of drugs and metabolites in plasma by high-throughput liquid chromatography tandem mass spectrometry (HTLC-MS/MS), we have developed a simple, sensitive and reliable method for simultaneous quantification of loratadine and desloratadine in human plasma. We have performed on-line coupling of extraction with Cyclone P 50 mm x 0.5 mm 50 microm HTLC column and chromatographic separation is performed with Zorbax XDB C18 50 mm x 2.1 mm 5 microm, followed by quantification with mass detector. The method is validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. A marked improvement in sample throughput efficiency is realized with this method and the proposed method will be useful for pharmacokinetic and/or bioequivalence studies.