Purification Platform Research Articles

Improved protein-A chromatography for monoclonal antibody purification

Therapeutic monoclonal antibodies become the major product class within the biopharmaceutical market. Protein A as the first capture step is still dominant in current platforms for purification of monoclonal antibodies. In this study, we developed a new antibody harvest process that incorporates acidic treatment of cell harvest, demonstrating high process yield, improved clearance of host cell associated contaminants, like non-histone host cell protein, histone, DNA and heteroaggregates. Host protein contamination was reduced about 10-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Turbidity increase of eluted IgG upon pH neutralization was nearly eliminated. Residual levels of impurities in the protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. The mechanism of host cell associated contaminants removal during acidic treatment was also explored. After a polishing step by Capto adhere, host cell protein was reduced to less than 5 ppm, DNA less than 1 ppb, histone to undetectable level, heteroaggregates less than 0.01% with total IgG recovery around 87%. This efficient process can be easily integrated into current IgG purification platforms, and may overcome downstream processing challenges.

An integrated inertial microfluidic vortex sorter for tunable sorting and purification of cells

Sorting of target cells from complex cellular samples into a high-purity product is challenging yet essential for downstream cell biology research and clinical diagnostics. Inertial microfluidics is an emerging technology attracting a lot of interest for passive and label-free sorting of cells with high throughput. Here, we introduce an inertial microfluidic device based on our vortex sorting platform for continuous size-based double sorting and purification of the larger target cells from the smaller background cells. Our device uses a microscale chamber with three outlets as a sorting unit, and integrates it into a specific topology to enable double sorting and purification functionalities. With properly designed fluidic resistance network and optimized flow conditions, we demonstrated continuous sorting of spiked human cancer stem-like cells from human blood with >90% efficiency and >1,500× enhanced purity, as well as removal of red blood cells with ~99.97% efficiency. We envision this integrated vortex-aided sorter can serve as a viable tool for size-based sorting of large target cells from complex cellular samples. Furthermore, this vortex-aided sorting platform can be integrated into more sophisticated topology with versatile functions for other cell sorting applications.

A robust robotic high-throughput antibody purification platform

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development.Miniaturization and automation of chromatography using micro columns (RoboColumns® from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO® from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH.The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A—desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.

Improved urine samples collection for DNA/RNA purification and its application in bladder cancer detection.

e18199 Background: Urine represents an extremely important body fluid for the sensitive and non-invasive detections of multiple diseases including urinary tract infections and cancers. Recently, urine nucleic acids based biomarkers has gained popularity as the preferred diagnostic methods. However, DNA and RNA in urine are prone to degradation and conventional urine storage (i.e. sample freezing and the addition of additives) have limited positive impacts on the nucleic acids quality. Therefore, to ensure the robust urinalysis, a well-standardized procedure of urine sample collection, storage, and nucleic acid purification should be developed and streamlined. Methods: We developed a novel technology, Urine Conditioning Buffer (UCB) which effectively preserves total DNA and RNA in urine samples for at least 1 month at ambient temperatures, abating the high costs of cold chain. Importantly, UCB inhibits microbial growth, reducing the risk of biohazard exposure. Additionally, the combination of UCB and Quick-DNA Urine Kit (Zymo Research) allows the isolation of either cellular, cell-free, or total DNA from urine samples. Results: We further validated our technology by conducting a pre-clinical study. The DNA methylation level of the 13 unique bladder cancer biomarkers (developed at Zymo Research) was analyzed in a cohort of 53 individuals (33 bladder cancer patients and 20 healthy individuals). Collected urine samples were preserved in UCB and total urine DNA was isolated using Quick-DNA Urine Kit. Urine DNA was analyzed in parallel using two different approaches: a bisulfite conversion based assay and a non-bisulfite based assay. Despite the methodology differences, both assays perfectly discriminated bladder cancer from control samples. This study indicates that UCBpreserves the urine DNA integrity, a prerequisite for the successful urine DNA and RNA based testing. Conclusions: Here, we present a successful application of our unique technology in bladder cancer diagnosis, which is the 5th most common cancer in US. Furthermore, it demonstrates that we have developed a total solution for urine based molecular testing covering sample collection, stabilization, purification, and detection platform.

460. Purification of AAV8 Using a Prototype Affinity Resin Compatible with cGMP Manufacturing

Affinity interaction based resins have long been the foundation for various purification platforms of biological molecules. For the purification of rAAV, the affinity resin AVB Sepharose HP (AVB) has been used to successfully purify a number of different serotypes. However, to date the use of AVB for the purification of the AAV8 serotype has been relatively unsuccessful due in part to modest binding capacity. In this work we investigate the use of a new, prototype affinity resin for the purification of AAV8 from crude feed streams. Performance of the AVB and prototype resins were directly compared for the purification of selected AAV8 feed streams. Differences in capacities, loading steps, wash step tolerances and elution steps will be discussed. In addition, the resulting performance of purifying AAV8 using the prototype resin is directly compared against the best case performance of the AVB resin purifying a different serotype more commonly used on AVB. Finally, the scale-up use of the prototype resin from bench to manufacturing will be discussed. These results demonstrate that the prototype affinity resin is a viable option for the scaleable, chromatographic purification of AAV8 from crude harvest streams.

ChemInform Abstract: A High‐Throughput Synthesis of 1,2,4‐Oxadiazole and 1,2,4‐Triazole Libraries in a Continuous Flow Reactor.

AbstractThe heterocyclization involves first a low‐temperature peptide coupling of various carboxylic acids and hydroxyamidines, hydrazonamides, or pyridyl hydrazides followed by a high‐temperature cyclization in a continuous flow process using a fully integrated synthesis and purification platform.

Application of the Protein Maker as a platform purification system for therapeutic antibody research and development

Within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mAbs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we describe specific applications and associated workflows of the Protein Maker liquid handling system utilized in both of these contexts. To meet the requirements for various in vitro assays, for the identification and validation of new therapeutic targets, small quantities of large numbers of purified antibodies or antibody fragments are often required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various wash buffers is an example of how parallelized protein purification can be leveraged to improve a process development outcome. Stability testing under various conditions of in-process intermediates, as an example, the mAb product from a clarified harvest, requires parallelized protein purification to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid scale platform purification or for process development applications to generate the necessary purified protein samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a significant reduction in time and cost per sample compared to serial purification using a traditional FPLC system. By combining the Protein Maker purification system with a TECAN Freedom EVO liquid handler for automated buffer exchange we have created a new, integrated platform for a variety of protein purification and process development applications.

Rational development of two flowthrough purification strategies for adenovirus type 5 and retro virus-like particles

We report on the rational design and implementation of flowthrough (FT) platforms for purification of virus vectors (VVs) and virus-like particles (VLPs), combining anion-exchange polyallylamine membranes (Sartobind STIC) and core–shell octylamine resins (CaptoCore 700). In one configuration, the VV bulk is concentrated and conditioned with appropriate buffer in a ultra/diafiltration (UF/DF) unit prior to injection into the STIC chromatography membrane. The FT pool and an intermediate cut of the elution pool of the STIC membrane are admixed and directed to a second UF/DF. Finally, the retentate is injected into a CC700 packed bed adsorber where the purified VVs are collected in the FT pool, whereas the residual amount of DNA and host cell protein (HCP) are discarded in the eluate. The experimental recovery achieved with this downstream processing (DSP) platform is close to 100%, the DNA clearance is roughly a 4-log reduction, and the HCP level is reduced by 5 logs. The platform developed for VLP purification is simpler than the previous one, as the STIC membrane adsorber and CC700 bed are connected in series with no UF/DF unit in between. Experimentally, the FT scheme for VLP purification gave a recovery yield of 45% in the chromatography train; the experimental log reduction of DNA and HCP were 2.0 and 3.5, respectively. These results are in line with other purification strategies in the specific field of enveloped VLPs. Both DSP platforms were successfully developed from an initial design space of the binding of the major contaminant (DNA) to the two ligands, determined by surface plasmon resonance, which was subsequently scaled up and confirmed experimentally.

Automated small‐scale protein purification and analysis for accelerated development of protein therapeutics

Small‐scale protein purification presents opportunities for accelerated process development of biotherapeutic molecules. Miniaturization of purification conditions reduces time and allows for parallel processing of samples, thus offering increased statistical significance and greater breadth of variables. The ability of the miniaturized platform to be predictive of larger scale purification schemes is of critical importance. The PerkinElmer JANUS BioTx Pro and Pro‐Plus workstations were developed as intuitive, flexible, and automated devices capable of performing parallel small‐scale analytical protein purification. Preprogrammed methods automate a variety of commercially available ion exchange and affinity chromatography solutions, including miniaturized chromatography columns, resin‐packed pipette tips, and resin‐filled microtiter vacuum filtration plates. Here, we present a comparison of microscale chromatography versus standard fast protein LC (FPLC) methods for process optimization. In this study, we evaluated the capabilities of the JANUS BioTx Pro‐Plus robotic platform for miniaturized chromatographic purification of proteins with the GE ӒKTA Express system. We were able to demonstrate predictive analysis similar to that of larger scale purification platforms, while offering advantages in speed and number of samples processed. This approach is predictive of scale‐up conditions, resulting in shorter biotherapeutic development cycles and less consumed material than traditional FPLC methods, thus reducing time‐to‐market from discovery to manufacturing.

Development of an aptamer-based affinity purification method for vascular endothelial growth factor

Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF) was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats.

For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode.

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow-rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log10 of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach.

Enveloped virus flocculation and removal in osmolyte solutions

Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2μm microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, this method has the potential to be a future platform purification process for vaccines.

A generic magnetic microsphere platform with "clickable" ligands for purification and immobilization of targeted proteins.

While much effort has been made to prepare magnetic microspheres (MMs) with surface moieties that bind to affinity tags or fusion partners of interest in the recombinant proteins, it remains a challenge to develop a generic platform that is capable of incorporating a variety of capture ligands by a simple chemistry. Herein, we developed core-shell structured magnetic microspheres with a high magnetic susceptibility and a low nonspecific protein adsorption. Surface functionalization of these MMs with azide groups facilitates covalent attachment of alkynylated ligands on their surfaces by "click" chemistry and creates a versatile platform for selective purification and immobilization of recombinant proteins carrying corresponding affinity tags. The general applicability of the approach was demonstrated in incorporating four widely used affinity ligands with different reactive groups (-CHO, -SH, -COOH, and -NH2) onto the MMs platform for purification and immobilization of targeted proteins. The azide-functionalized MMs would be applicable for a variety of ligands and substrates that are amenable to alkynylation modification.

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance.

Automated centrifugal-microfluidic platform for DNA purification using laser burst valve and coriolis effect.

We report a fully automated DNA purification platform with a micropored membrane in the channel utilizing centrifugal microfluidics on a lab-on-a-disc (LOD). The microfluidic flow in the LOD, into which the reagents are injected for DNA purification, is controlled by a single motor and laser burst valve. The sample and reagents pass successively through the micropored membrane in the channel when each laser burst valve is opened. The Coriolis effect is used by rotating the LOD bi-directionally to increase the purity of the DNA, thereby preventing the mixing of the waste and elution solutions. The total process from the lysed sample injection into the LOD to obtaining the purified DNA was finished within 7 min with only one manual step. The experimental result for Salmonella shows that the proposed microfluidic platform is comparable to the existing devices in terms of the purity and yield of DNA.

Large-scale crystallization of proteins for purification and formulation.

Since about 170years, salts were used to create supersaturated solutions and crystallize proteins. The dehydrating effect of salts as well as their kosmotropic or chaotropic character was revealed. Even the suitability of organic solvents for crystallization was already recognized. Interestingly, what was performed during the early times is still practiced today. A lot of effort was put into understanding the underlying physico-chemical interaction mechanisms leading to protein crystallization. However, it was understood that already the solvation of proteins is a highly complex process not to mention the intricate interrelation of electrostatic and hydrophobic interactions taking place. Although many basic questions are still unanswered, preparative protein crystallization was attempted as illustrated in the presented case studies. Due to the highly variable nature of crystallization, individual design of the crystallization process is needed in every single case. It was shown that preparative crystallization from impure protein solutions as a capture step is possible after applying adequate pre-treatment procedures like precipitation or extraction. Protein crystallization can replace one or more chromatography steps. It was further shown that crystallization can serve as an attractive alternative means for formulation of therapeutic proteins. Crystalline proteins can offer enhanced purity and enable highly concentrated doses of the active ingredient. Easy scalability of the proposed protein crystallization processes was shown using the maximum local energy dissipation as a suitable scale-up criterion. Molecular modeling and target-oriented protein engineering may allow protein crystallization to become part of a platform purification process in the near future.

Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG.

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.

Novel biocompatible and self-buffering ionic liquids for biopharmaceutical applications.

Antibodies obtained from egg yolk of immunized hens, immunoglobulin Y (IgY), are an alternative to the most focused mammal antibodies, because they can be obtained in higher titers by less invasive approaches. However, the production cost of high-quality IgY for large-scale applications remains higher than that of other drug therapies due to the lack of efficient purification methods. The search for new purification platforms is thus vital. The solution could be liquid-liquid extraction by using aqueous biphasic systems (ABS). Herein, we report the extraction and attempted purification of IgY from chicken egg yolk by using a new ABS composed of polymers and Good's buffer ionic liquids (GB-ILs). New self-buffering and biocompatible ILs based on the cholinium cation and anions derived from Good's buffers were synthesized and the self-buffering characteristics and toxicity were characterized. Moreover, when these GB-ILs are combined with PPG 400 (poly(propylene) glycol with a molecular weight of 400 g mol(-1)) to form ABS, extraction efficiencies, of the water-soluble fraction of proteins, ranging between 79 and 94% were achieved in a single step. Based on computational investigations, we also demonstrate that the preferential partitioning of IgY for the GB-IL-rich phase is dominated by hydrogen-bonding and van der Waals interactions.

Flexible Porous Zinc–Pyrazole–Adenine Framework for Hysteretic Sorption of Light Hydrocarbons

One porous metal–organic framework based on mixed ligands, [Zn5(4-pca)4(ade)2(H2O)2]·(DMF)·4(H2O) (1; 4-H2pca = 4-pyrazolecarboxylic acid, Hade = adenine, DMF = N,N-dimenthylformamide), has been synthesized solvothermally and structurally characterized. Interestingly, the framework of 1 shows reversible flexibility, so that the adsorption behaviors of this activated sample exhibit the gate-opening effect in response to light hydrocarbons (C2H2, C2H4, C2H6, and C3H8). As a result, it could serve as an excellent platform for storage and purification of small hydrocarbons.