Protein Purification Process Research Articles

Apoplastic Expression of CARD1-ecto Domain in Nicotiana benthamiana and Purification from the Apoplastic Fluids.

The protein expression and purification process is an essential initial step for biochemical analysis of a protein of interest. Traditionally, heterologous protein expression systems (such as E. coli, yeast, insect cells, and cell-free) are employed for plant protein expression, although a plant expression system is often desirable for plant proteins, to ensure proper post-translational modifications. Here, we describe a method to express and purify the ectodomain of one of the leucine-rich repeat receptor-like kinase called CARD1/HPCA1, from Nicotiana benthamiana apoplastic fluid. First, we express His-tagged CARD1 ectodomain in the apoplastic space of N. benthamiana by the Agroinfiltration method. Then, we collect apoplastic fluids from the leaves and purify the His-tagged protein by Ni2+-affinity chromatography. In addition to plant-specific post-translational modifications, protein accumulated in the plant apoplastic space, rather than in the cytosolic space, should be kept under an oxidizing environment. Such an environment will help to maintain the property of intrinsic disulfide bonds in the protein of interest. Further, purification from the apoplastic fluids, rather than the total protein extract, will significantly reduce contaminants (for instance RuBisCO) during protein extraction, and simplify downstream processes. We envisage that our system will be useful for expressing various plant proteins, particularly the apoplastic or extracellular regions of membrane proteins.

Expert Roundtable: evolution in AAV process development: 2022 and beyond

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/Alejandro Becerra Headshot.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#5D538A;">Alejandro Becerra</h3><h5 class="author-title" style="color:#5D538A;">Principal Applications Scientist and Global Purification Technical Lead, Thermo Fisher Scientific</h5> Dr Alejandro Becerra is a Principal Applications Scientist and Global Purification Technical Lead. Alejandro has over 14 years of experience in downstream processing and customer support having worked as Purification Team Manager and other bioprocess engineering roles prior to joining Thermo Fisher Scientific in 2018. Dr Becerra is a subject matter expert in preparative chromatography with expertise in the development, optimization and scale-up of antibody, recombinant protein and viral vector purification processes. Alejandro holds a PhD in Chemical Engineering from Cornell University. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/Matthias Hebben copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#00A795;"><strong>Matthias Hebben</strong></h3><h5 class="author-title" style="color:#00A795;">Vice President of Technology Development, LogicBio Therapeutics</h5> Matthias Hebben has been serving as vice president of technology development at LogicBio Therapeutics since February 2019. In his role, he is leading the CMC efforts, including vector core, capsid optimization, process development, analytical development and clinical product manufacturing. Before that, he served as director of technology development and head of bioprocess development at Genethon for 6 years. Before that, he occupied several positions at Vivalis (Valneva), Intervet Shering Plough and Virbac. Matthias has a PhD in molecular biology and a MSc in bioprocess engineering </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><strong><strong><strong><img src="https://bioinsights.blob.core.windows.net/uploads/Mercaldi Headshot copy.jpg" data-image="1"></strong></strong></strong></div><div class="author-det"><h3 class="author-name" style="color:#007589;"><strong><strong>Michael Mercaldi</strong></strong></h3><h5 class="author-title" style="color:#007589;">Senior Director of Downstream Process Development, Homology Medicines</h5> Michael is the Senior Director of Downstream Process Development at Homology Medicines. He is responsible for leading the development of Homology’s purification and drug product manufacturing processes for their gene therapy and gene editing programs. He has held positions in process development throughout his career at MedImmune/AstraZeneca, Merrimack Pharmaceuticals and Codiak Biosciences before joining Homology. He holds a BS in Chemical Engineering from the Rensselaer Polytechnic Institute and a PhD in Biochemical Engineering from Tufts University. </div></div></div>

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli.

Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, simplify the recombinant protein purification process, and promote the screening of molecules that fail to remain soluble after tag removal. High yields of soluble target proteins have already been achieved using these protease co-expression systems. Herein, we review approaches for controlled intracellular processing systems tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular processing of recombinant proteins regarding system design features, advantages, and limitations of the various strategies.

Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens.

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

Soluble Expression, One-Step Purification and Characterization of Recombinant Human Growth Hormone Fused with ompA3 in Escherichia coli.

Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli. To increase the soluble expression of recombinant hGH with correct folding in E. coli. We constructed a new recombinant expression plasmid containing the coding sequence of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3) E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion sequence should contain hexahistidine-tag (His6) and enterokinase recognition sites (D4K). The effect of different expression conditions on recombinant hGH expression was optimized in flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified hGH was also evaluated by cell proliferation assay. High-level expression (800 μg/mL) was achieved by induction with 0.5 mM IPTG at 30°C for 10 hours. The purity of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show the highest activity at a concentration of 100 ng/mL. Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E. coli, and may lay a foundation for the production and application of hGH in the industry.

Quality control of purified proteins to improve data quality and reproducibility: results from a large-scale survey.

As the scientific community strives to make published results more transparent and reliable, it has become obvious that poor data reproducibility can often be attributed to insufficient quality control of experimental reagents. In this context, proteins and peptides reagents require much stricter quality controls than those routinely performed on them in a significant proportion of research laboratories. Members of the ARBRE-MOBIEU and the P4EU networks have combined their expertise to generate guidelines for the evaluation of purified proteins used in life sciences and medical trials. These networks, representing more than 150 laboratories specialized in protein production and/or protein molecular biophysics, have implemented such guidelines in their respective laboratories. Over a one-year period, the network members evaluated the contribution these guidelines made toward obtaining more productive, robust and reproducible research by correlating the applied quality controls to given samples with the reliability and reproducibility of the scientific data obtained using these samples in follow-up experiments. The results indicate that QC guideline implementation facilitates the optimization of the protein purification process and improves the reliability of downstream experiments. It seems, therefore, that investing in protein QC might be advantageous to all the stakeholders in life sciences (researchers, editors, and funding agencies alike), because this practice improves data veracity and minimizes loss of valuable time and resources. In the light of these conclusions, the network members suggest that the implementation of these simple QC guidelines should become minimal reporting practice in the publication of data derived from the use of protein and peptide reagents.

Fabrication and simulation studies of high-performance anionic sponge alginate beads for lysozyme separation

The large-scale applications of lysozyme in the pharmaceutical industry and food industry require more efficient and cost-effective techniques for its separation. This study present scalable methodology for development of highly porous chromatographic media using an in situ formed network of natural polysaccharide alginate (Alg) through using CaCO3 as solid porogen. The as-prepared porous (Alg) beads demonstrated porous structures with pore diameter 200 nm and 94% porosity. Porous (Alg) beads were functionalized via sodium 3-sulfobenzoate (SS) for lysozyme separation via electrostatic attraction. The optimized sulfonated-alginate beads (SS(8)-Alg(0.7)) exhibited excellent lysozyme adsorption capability of 600 mg g−1 within short adsorption time 6 h. More importantly, it shows a dynamic binding efficiency of 500 mg g−1 can be achieved only by the gravity of solution, which matches well with the demands of the high yield and energy conservation in the actual protein purification process. The (SS(8)-Alg(0.7)) beads exhibited high selectivity that enable it directly extract lysozyme from egg white with a relatively large capture capability of 300 mg g−1. Furthermore, the NFM displayed efficient chemical and physical stability even after 5 cycles without significant change in its porous nature or adsorption performance. Significantly, aiming to expand this efficient technique in industrial scale we apply the operating parameters using response surface methodology (RSM) as a modeling tool to optimize the process for further proteins molecules. The premise of this design is that the reusable sulfonate groups are covalently incorporated into the open porous beads structure and can be directly used in reliable separation applications through batch mode or under the driven pressure of gravity.

Rapid Surface Modification of Ultrafiltration Membranes for Enhanced Antifouling Properties.

In this work, several ultrafiltration (UF) membranes with enhanced antifouling properties were fabricated using a rapid and green surface modification method that was based on the plasma-enhanced chemical vapor deposition (PECVD). Two types of hydrophilic monomers—acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) were, respectively, deposited on the surface of a commercial UF membrane and the effects of plasma deposition time (i.e., 15 s, 30 s, 60 s, and 90 s) on the surface properties of the membrane were investigated. The modified membranes were then subjected to filtration using 2000 mg/L pepsin and bovine serum albumin (BSA) solutions as feed. Microscopic and spectroscopic analyses confirmed the successful deposition of AA and HEMA on the membrane surface and the decrease in water contact angle with increasing plasma deposition time strongly indicated the increase in surface hydrophilicity due to the considerable enrichment of the hydrophilic segment of AA and HEMA on the membrane surface. However, a prolonged plasma deposition time (>15 s) should be avoided as it led to the formation of a thicker coating layer that significantly reduced the membrane pure water flux with no significant change in the solute rejection rate. Upon 15-s plasma deposition, the AA-modified membrane recorded the pepsin and BSA rejections of 83.9% and 97.5%, respectively, while the HEMA-modified membrane rejected at least 98.5% for both pepsin and BSA. Compared to the control membrane, the AA-modified and HEMA-modified membranes also showed a lower degree of flux decline and better flux recovery rate (>90%), suggesting that the membrane antifouling properties were improved and most of the fouling was reversible and could be removed via simple water cleaning process. We demonstrated in this work that the PECVD technique is a promising surface modification method that could be employed to rapidly improve membrane surface hydrophilicity (15 s) for the enhanced protein purification process without using any organic solvent during the plasma modification process.

Catalytically Active Bioseparation Resin Utilizing a Covalent Intermediate for Tagless Protein Purification.

Common protein purification processes rely on noncovalent affinity binding of the target protein to resin via fusion tags, which has limitations such as leakage of target during the removal of impurities, restricting stringency of wash conditions, and often requires postpurification removal of tags. Here, we developed the Catch, Hold, and Release (C-H-R) method by bioengineering a single-use resin composed of polyhydroxybutyrate beads codisplaying two SpyCatcher (SpyC) domains and Sortase A (SrtA). This core-shell resin was assembled by engineered Escherichia coli. SpyTagged (SpyT) targets with a SrtA cleavage site after SpyT were specifically ligated to SpyC, while inducible SrtA catalyzed the release of the pure tagless target. SpyT remained covalently bound to the resin. Three diverse proteins were isolated from complex mixtures with high purity and yields. The C-H-R bioseparation method uses an innovative resin engaging a covalent link to the target and an inducible proximal SrtA as a protein purification concept.

Improvements of a high-throughput protein purification process using a calcium-dependent setup

The Human Secretome Project aims to produce and purify all human secreted proteins as full-length. In order to enable this, a robust, gentle and effective purification process is needed, where multiple proteins can be purified in parallel. For this reason, a purification system based on a Protein C-tag and the HPC4 antibody with high affinity to the tag was chosen for purification. The strong binding between the tag and the antibody is specific and calcium-dependent, which allows for mild elution with EDTA. Presented here is a study comparing different protein purification base matrices coupled with the HPC4 antibody, aiming to increase the yield of purified protein and reduce the time for purification. Among the different tested matrices, Capto XP showed a high coupling degree and increased the amount of eluted protein as compared to the control matrix. By moving from batch incubation to direct sample loading and by performing the purification on the ÄKTAxpress, an automated protein purification process and a high reduction of hands-on sample handling was achieved. This new method also integrates the desalting step in the purification process, and the time for purification and analysis of each sample was decreased from five to three days. Moreover, a new mild method for matrix regeneration was developed using 50 mM EDTA pH 7.5 instead of 0.1 M glycine pH 2. This method was proven to be efficient for regeneration while maintaining the column binding performance even after nine rounds of regeneration.

Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system

Protein purification processes in basic research using ÄKTA™ liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS™ autosampler and a modular sample in-line dilution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion. The chromatographic setup enabled purification of a large array of cytosolic and membrane proteins from small-scale expression cultures produced in insect cells necessary to develop and optimize isotope-labeling strategies for nuclear magnetic resonance spectroscopy applications, resulting in a reduction in experiment time of about 20% per run for both cytosolic and membrane protein purification schemes. However, when queuing multiple samples the throughput increased by 66% and 75%, respectively. In addition, a novel system configuration is presented, where two column valves can be operated independently. This allows for the design of purification loops to increase purity of the target protein.

Understanding adsorption behavior of α-chymotrypsin onto cation exchanger using all-atom molecular dynamics simulations

The interest for a better understanding of ion-exchange mechanisms at the atomic level has strongly increased over the past decades. Indeed, molecular-level information about physico-chemical mechanisms could help optimizing chromatographic processes for protein purification, which are sub-optimized due to the lack of predictive models. A promising approach is based on the use of Molecular Dynamics (MD) simulations to study local phenomena inside adsorbents which can then be challenged against experimental results. In this work, macroscopic experimental data, consisting in the ion-exchange uptake of α-chymotrypsin onto SP Sepharose FF, have been compared to the adsorption behavior predicted by MD simulations. The chromatographic surface, represented as a uniform distribution of ligands with a counterion layer, in the presence of the protein was modeled using all-atom representation. The SMA formalism was used to describe single adsorption isotherms and to relate macroscopic observations with molecular simulations. Two SMA parameters based on physical principles, the characteristic charge n and the steric factor σ, have been estimated by both experiments and MD simulations. At pH 5 and NaCl concentration of 100 mM, our study shows a fairly good agreement between both results, especially for the characteristic charge. It is shown that the steric factor calculation is strongly dependent on the ligand density on the adsorbent surface, whose value must be carefully determined in order to obtain reliable predictions. In addition, four binding patches were identified as being involved in the adsorption, which have been confirmed through binding free energy calculations.

High-Throughput Quantitative Top-Down Proteomics: Histone H4.

Proteins physiologically exist as "proteoforms" that arise from one gene and acquire additional function by post-translational modifications (PTM). When multiple PTMs coexist on single protein molecules, top-down proteomics becomes the only feasible method of characterization; however, most top-down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that top-down proteomics can be quantitative, reproducible, sensitive, and high throughput. The proteoforms of histone H4 are well studied both as a challenging proteoform identification problem and due to their essential role in the regulation of all eukaryotic DNA-templated processes. Much of histone H4's function is obfuscated from prevailing methods due to combinatorial mechanisms. Starting from cells or tissues, after an optimized protein purification process, the H4 proteoforms are physically separated by on-line C3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. We achieve more than 30 replicates from a single 35-mm tissue culture dish by loading 55 ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day. We achieve reproducible quantitation (average biological Pearson correlations of 0.89) of hundreds of proteoforms (about 200-300) over almost six orders of magnitude and an estimated LLoQ of 0.001% abundance. We demonstrate the capacity of the method to precisely measure well-established changes with sodium butyrate treatment of SUM159 cells. We show that the data produced by a quantitative top-down method can be amenable to parametric statistical comparisons and is capable of delineating relevant biological changes at the full proteoform level.

A direct comparison between membrane adsorber and packed column chromatography performance

The purpose of this work was to compare side by side the performance of packed bed and membrane chromatography adsorption processes for protein purification. The comparison was performed using anion exchange media with the same ligand immobilized on the adsorbing surface, namely the strong Q quaternary ammonium group, R-CH2-N+-(CH3)3, and bovine serum albumin (BSA) as a model protein. In addition, the stationary phase volume was held constant for each geometry (3 mL) and runs were executed using the same mobile phase superficial velocity. As expected, the packed bed column showed higher equilibrium binding of BSA at 66.9 mg/mL versus 43.04 mg/mL for the membrane adsorber. Dynamic binding capacities were also higher in the packed bed; for example, at 97.5 cm/h, a capacity of 62.8 mg/mL was measured for the packed bed versus 20.7 mg/mL for the membrane adsorber. The higher equilibrium and dynamic capacities of the packed bed are likely due to the higher surface area per unit volume of the resin. However, the maximum productivity for the membrane adsorber was 111 mg/(mL h), a value that was 3.3 times higher than the one of the packed column. The bed utilization - defined as the ratio of the dynamic binding capacity at 10% breakthrough to the saturation binding capacity - was also higher for the packed column at long residence times and lower at short residence times confirming the better performance of membrane chromatography at high flow rates.

Genetically Encoded Residue-Selective Photo-Crosslinker to Capture Protein-Protein Interactions in Living Cells

Summary The genetically encoded non-selective photo-crosslinkers suffer from unpredictable crosslinking sites and false positive interacting proteins, thus, the identification of protein interactions and mapping accurate interaction interfaces remain largely elusive in living systems. Here, we report genetically encoded, residue-selective protein photo-crosslinker enabling covalent crosslinking of the interacting proteins with proximal lysine, upon UV light activation in vitro and in living cells. Using this photo-crosslinker, we demonstrate a crosslinking strategy to capture protein-protein interactions with residue selectivity in a temporal-resolved manner. In addition, this crosslinking strategy produces the lysine-selective crosslinking site and predictable crosslinked peptide, which serve as the direct evidence for protein-protein interactions and facilitate mass spectrometry analysis. More importantly, this photo-crosslinker enables the capture of elusive enzyme-substrate interaction with directly interacting lysine and validation of acetylation site of the substrate. This strategy represents higher spatiotemporal resolution, accuracy, and reliability for investigating dynamic, transient, and weak protein-protein interactions in living systems.

Development of BioRad NGC and GE ÄKTA pure systems for highly automated three column protein purification employing tandem affinity, buffer exchange and size exclusion chromatography

Affinity purification, such as Protein A (ProA) followed by size exclusion chromatography (SEC) remains a popular method to obtain research scale proteins. With the need for higher throughput protein production increasing for discovery research, there is substantial interest in the automation of complex protein purification processes, which often start with a ProA step followed by SEC. However, the harsh elution conditions from ProA based chromatography can destabilize some proteins resulting in particulates, which in turn can cause column fouling and potential cross-contamination of subsequent purifications. We modified both Bio Rad NGC and ÄKTA Pure systems to run a three-column process (ProA to buffer exchange to SEC) enabling automated tandem affinity to SEC purification while minimizing the risk of SEC column fouling and subsequent cross-contamination. The intervening buffer exchange column, unlike the final SEC column, can be rapidly regenerated using harsh methods between runs, and these automated systems are capable of processing up to six samples per day without user intervention.

First comprehensive view on a magnetic separation based protein purification processes: From process development to cleaning validation of a GMP-ready magnetic separator.

Magnetic separation processes are known as integrated bioanalytical protein purification method since decades and are well described. However, use of magnetic separation processes in a regulated industrial production environment has been prevented by the lack of suitable process equipment and prejudice against the productivity of the process and its qualification for cleaning-in-place operation. With the aim of overcoming this prejudice, a comprehensive process development approach is presented, based on a GMP-compliant magnetic separator, including an optimization of the batch adsorption process, implementation into a technical-scale, and the development and validation of cleaning routines for the device. By the implementation of a two-step counter-current binding process, it was possible to raise the yields of the magnetic separation process even for very low concentrated targets in a vast surplus of competing proteins, like the hormone equine chorionic gonadotropin in serum, from 74% to over 95%. For the validation of the cleaning process, a direct surface swabbing method combined with a total organic carbon analysis was established for the determination of two model contaminants. The cleanability of the process equipment was proven for both model contaminants by reliably meeting the 10ppm criteria.

A High-Throughput System for Transient and Stable Protein Production in Mammalian Cells.

Recombinant protein expression and purification is an essential component of biomedical research and drug discovery. Advances in automation and laboratory robotics have enabled the development of highly parallel and rapid processes for cell culture and protein expression, purification, and analysis. Human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells have emerged as the standard host cell workhorses for producing recombinant secreted mammalian proteins by using both transient and stable production strategies. In this chapter we describe a fully automated custom platform, Protein Expression and Purification Platform (PEPP), used for transient protein production from HEK cells and stable protein production from CHO cells. Central to PEPP operation is a suite of custom robotic and instrumentation platforms designed and built at GNF, custom cell culture ware, and custom scheduling software referred to as Runtime. The PEPP platform enables cost-effective, facile, consistent production of proteins at quantities and quality useful for early stage drug discovery tasks such as screening, bioassays, protein engineering, and analytics.

Endotoxin reduction in protein solutions using octyl β-D-1-thioglucopyranoside wash on chromatography media

Endotoxins are complex molecules and one of the most challenging impurities requiring separation in biopharmaceutical protein purification processes. Usually these contaminants are cleared during the downstream process, but if endotoxin interacts with the target protein it becomes difficult to remove. In the present study we identified a detergent, octyl-β-D-1-thioglucopyranoside (OTG), that disrupted endotoxin-protein interactions. The integration of this detergent into washes on several chromatography media was demonstrated to provide a separation tool for decreasing endotoxin from target proteins. This study also examined the mechanism of OTG endotoxin-protein disruption through phase modification incubation and chromatographic studies. The non-ionic OTG wash was shown to break both hydrophobic and electrostatic interactions between the endotoxin and protein. This mechanism contrasts with the breaking of hydrophobic interactions by washing with known endotoxin decreasing Triton X-100 detergent. The difference in mechanisms likely results in the ability of OTG to decrease endotoxin to levels less than those resulting from a detergent wash such as Triton X-100. Finally, we show the impact of the OTG endotoxin removal tool on the biopharmaceutical industry. While maintaining monomer purity and activity levels, endotoxin removal from a fusion protein allowed for decreased background levels in a T cell functional assay. The lowered baseline of T cell responses allowed for more effective detection of molecular interaction with the cells. The detergent wash can be used to both decrease the overall level of endotoxin in a purified protein solution and to enable more effective screening of lead candidate molecules.

Single pass diafiltration integrated into a fully continuous mAb purification process.

The concept of continuous manufacturing has gained significant interest from the biopharmaceutical industry over the past several years. Benefits include increased manufacturing productivity, improved quality control, reduction in plant footprint, and more flexible management of facility capacity. There are several technologies currently available that enable continuous processing for chromatography and ultrafiltration. However, a single pass diafiltration design that meets the required small molecule clearance and has been integrated into a fully continuous monoclonal antibody purification process has not been previously published. Here, the theory and design of a 3-stage single pass diafiltration step is presented. Buffer exchange greater than 99.75% was experimentally demonstrated. Several critical design aspects were incorporated to minimize system complexity and reduce the buffer volume requirements. Lastly, single pass diafiltration was demonstrated in a pilot scale continuous process with uninterrupted flow from the bioreactor through the formulation step. This work illustrates the feasibility of incorporating a single pass diafiltration step into an end-to-end continuous protein purification process.