High-performance countercurrent membrane purification for host cell protein removal from monoclonal antibody products

The transition to continuous biomanufacturing has led to renewed interest in alternative approaches for downstream processing of monoclonal antibody (mAb) products. In this study, we examined the potential of using high-performance countercurrent membrane purification (HPCMP) for the removal of host cell proteins (HCPs) derived from Chinese Hamster Ovary cells in the purification of a mAb. Initial studies used several model proteins to identify appropriate operating conditions for the hollow fiber membrane modules. HPCMP was then used for mAb purification, with mAb yield >95% and more than 100-fold reduction in HCP. Stable operation was maintained for 48 h for feeds that were first prefiltered through the 3M^TM Harvest RC chromatographic clarifier to remove DNA and other foulants. In addition, the Process Mass Intensity for HPCMP can be much less than that for alternative HCP separation processes. These results highlight the potential of using HPCMP as part of a fully continuous mAb production process.     

Unstable proteins: how to subject them to chromatographic separations for purification procedures

The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.     

A new integrated membrane filtration and chromatographic device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.     

Evaluation of Escherichia coli proteins that burden nonaffinity-based chromatography as a potential strategy for improved purification performance

Escherichia coli is a favored host for rapid, scalable expression of recombinant proteins for academic, commercial, or therapeutic use. To maximize its economic advantages, however, it must be coupled with robust downstream processes. Affinity chromatography methods are unrivaled in their selectivity, easily resolving target proteins from crude lysates, but they come with a significant cost. Reported in this study are preliminary efforts to integrate downstream separation with upstream host design by evaluating co-eluting host proteins that most severely burden two different nonaffinity-based column processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of green fluorescent protein (GFP) by hydrophobic interaction and anion exchange chromatography. Ribosomal protein L25 dominated non-target binding of polyarginine-tagged GFP on cation exchange resin. Implications for genetic knockout or site-directed mutagenesis resulting in diminished column retention are discussed for these and other identified contaminants.     

Protein fractionation using fast flow immobilized metal chelate affinity membranes

A new group-specific affinity membrane using metal chelates as ligands and inorganic glass hollow fiber microfiltration membranes as support matrices is developed and tested. The study focused on developing the optimum activation and coupling procedures to bind the chelating agent (iminodiacetic acid, IDA) to the surface of the microporous glass hollow fiber membrane and testing the resultant affinity membrane. Starting with three different glass surfaces, five modification reactions were evaluated. All the modified "active surfaces" were first tested for their protein adsorptive properties in batch mode with suspended microporous glass grains using model proteins with known binding characteristics with Cu-IDA systems. The metal loading capacities of the surfaces exhibiting favorable fractionation were then measured by atomic absorption spectroscopy.The results were compared with the results obtained with a commercial material used in immobilized metal affinity column chromatography. The protein binding characteristics of the hollow fiber affinity membranes were also evaluated under conditions of convective flow. This was performed by flowing single solute protein solutions through the microporous membrane at different flow rates. These results were then used to estimate the optimum loading and elution times for the process. A mathematical model incorporating radial diffusion was solved using a finite difference discretization method. Comparison between model predictions and experimental results was performed for four different proteins at one flow rate. These results suggested that the kinetics of adsorption was concentration dependent. Finally, the hollow fiber affinity membranes were challenged with two component mixtures to test their ability to fractionate mixed protein solutions. Efficient separation and good purity were obtained.The results presented here represent the development of a new fast flow affinity membrane process-immobilized metal affinity membranes (IMAM). (c) 1994 John Wiley & Sons, Inc.     

Protein loading,elution, and resolution behavior in a novel device that integrates ultrafiltration and chromatographic separation

Hollow fiber membranes and chromatographic resin beads are commonly employed in a variety of bioseparation processes. A new class of integrated separation devices is being studied in which the shell side of a hollow fiber device is filled with adsorbents/chromatographic resin beads. Such devices and the corresponding separation methods integrate feed broth clarification by the microfiltration/ultrafiltration membrane with bioproduct purification by the shell-side resin beads either as an adsorbent or as beads in elution chromatography. A mathematical model has been developed for the prediction of the chromatographic behavior of such an integrated device. Simulations have been done to study the effects of axial dispersion, feed flow rate, water permeation rate, fiber packing density, and void fraction. Numerical solutions were obtained by solving the governing equations. This model can reasonably describe the concentration profiles as well as the breakthrough and elution behaviors in the integrated device.     

Rapid extraction and purification of lumbrokinase from <Emphasis Type="Italic">Lumbricus rubellus</Emphasis> using a hollow fiber membrane and size exclusion chromatography

Objectives

To develop a purification process using hollow fiber membrane separation combined with size exclusion chromatography for the extraction of lumbrokinase from earthworms (Lumbricus rubellus).

Results

To extract the protein, the earthworms were first homogenized for 10 min, to produce ultrafine particles. Polyether sulfone hollow fiber membranes with MW cut offs of 50 and 6 kDa were used for initial purification of the crude extract. Further purification was carried out on a Sephadex G-75 column, and yielded three fractions of high purity protein. One of these fractions showed fibrinolytic activity.

Conclusions

Three samples of high purity protein were obtained and one protein (LK1) showed strong fibrinolytic activity. The method has higher purification efficiency in comparison with existing methods.

     

Downstream process design for Gag HIV-1 based virus-like particles

Virus-like particles-based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus-like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus-like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 10¹⁰ virus-like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV-1 Gag-eGFP virus-like particles suitable for scale-up.     

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.     

Simultaneous ultrafiltration and affinity sorptive separation of proteins in a hollow fiber membrane module

A protein separation scheme combining affinity or ion exchange sorption with hollow fiber cross-flow filtration is described. Sorptive gel particles were loaded into the shell side of a hollow fiber membrane module. In the adsorption step, crude protein mixtures were passed through the lumen and permeating proteins passed through the membrane to bind on the gel particles in the shell. During elution, a buffer of adequate ionic strength to desorb the bound proteins was passed through the lumen and permeated through the shell. The eluant was then collected at the outlet to the shell of the hollow fiber module. The concept is illustrated by two examples: the purification of butyrylcholinesterase (EC 3.1.1.7) from raw horse serum using the affinity gel procainamide-Sepharose as the packing and the separation of carboxylesterase (EC 3.1.1.1) from beef liver homogenate using DEAE-Sephadex as the packing. The technique has the advantage of high volumetric throughputs typical of hollow fiber membrane modules as well as the high capacity characteristic of chromatographic packings. In addition, cross-flow filtration of particulates, agglomerates, and debris in passing protein from lumen to shell side can help eliminate the need for extensive pretreatment.     

A novel downstream process for 1,3-propanediol from glycerol-based fermentation

In this paper, a downstream process for purification of 1,3-propanediol from glycerol-based fermentation broth was investigated. The purification of 1,3-propanediol from fermentation broth was achieved by a process combining microfiltration, charcoal treatment, vacuum distillation, and silica gel chromatography. The broth was first filtered through hollow fiber cartridge, wherein 98.7% of biomass was removed. Soluble proteins and other color impurities in the broth were removed by the use of activated charcoal at optimal concentration of 30 g l⁻¹ where the soluble proteins in the broth decreased to 0.1 g l⁻¹ (96.0% protein loss). The obtained broth when concentrated by vacuum distillation resulted in the crystallization of inorganic salts. Subsequently, 1,3-propanediol was purified by gradient chromatography using silica gel as a stationary phase and mixture of chloroform and methanol as a mobile phase. Finally, with the optimal flow rate of 10 ml min⁻¹ and loading amount of 80 ml, the yield of 1,3-propanediol achieved was 89%. The overall yield of 1,3-propanediol using the proposed procedure was 75.47%. The developed method was found to be a simple, rapid, and efficient procedure for the purification of 1,3-propanediol from fermentation broth.     

Hollow fiber countercurrent dialysis for continuous buffer exchange of high-value biotherapeutics

Buffer exchange, desalting, and formulation of high-value biotherapeutics are currently performed using batch diafiltration (DF); however, this type of tangential flow filtration process may be difficult to implement as part of a fully continuous biomanufacturing process. The objective of this study was to explore the potential of using countercurrent dialysis for continuous protein formulation and buffer exchange. Experiments were performed using concentrated solutions of immunoglobuin G (IgG) with commercially available hollow fiber dialyzers having 1.5 and 1.8 m² membrane surface area. More than 99.9% buffer exchange was obtained over a range of conditions, as determined from the removal of a model impurity (vitamin B₁₂). The dialyzers were able to process more than 0.5 kg of IgG per day in an easily scalable low-cost process. In addition, buffer requirements were less than 0.02 L of buffer per gram IgG, which is several times less than that used in current batch DF processes. These results clearly demonstrate the potential of using low-cost hollow fiber dialyzers for buffer exchange and product formulation in continuous bioprocessing. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2763, 2019.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Is there a rational method to purify proteins? From expert systems to proteomics

The purification of recombinant proteins for therapeutic or analytical applications requires the use of several chromatographic steps in order to achieve a high level of purity. A range of techniques is available such as anion and cation exchange chromatography, which can be carried out at different pHs, and hence used at different steps, hydrophobic interaction chromatography, gel filtration and affinity chromatography. Evidently when confronted with a complex mixture of partially unknown proteins or a clarified cell extract there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98, 99.5 or 99.9%). In this review we will show how an initial "proteomic" characterization of the complex initial mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a maximum level of purity with a minimum number of steps. The chosen methodology was implemented in a computer based expert system. The first algorithm developed was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants. The second algorithm developed was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The successful application of the expert system approach, based on an initial proteomic characterization, to the practical cases of protein mixtures and clarified fermentation supernatant is presented and discussed. The purification strategy proposed was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with an "unknown" supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase. The system was robust to errors <10% which is the range that can be found in the experimental determination of the properties in the database of product and contaminants. On the other hand, the system was sensitive both to larger variations (>20%) in the properties of the contaminant database and the protein product and to variations in one protein property (e.g. hydrophobicity).     

Enhanced purification of plasmid DNA using Q-Sepharose by modulation of alcohol concentrations

Ion-exchange chromatography is one of the most commonly used methods for plasmid preparation. In this study a modified method was used to purify plasmid from bacterial lysate using Q-Sepharose. Incorporation of alcohols into the washing buffers enhanced the separation of plasmid from RNA and proteins. The use of isopropanol and ethanol achieved a high yield and purity whereas the use of methanol failed to improve the plasmid purification using Q-Sepharose by batch adsorption-desorption. Stepwise elution containing various concentrations of isopropanol and NaCl was used in preparative chromatography to enhance the plasmid purification. The same stepwise elution was applied to the chromatography columns packed with 0.5, 20, and 200 ml of Q-Sepharose for plasmid purification from 7.5, 300, and 3000 ml bacterial broth, respectively. Complete separation of DNA from RNA and proteins was achieved under gravity flow by modulation of the alcohol concentrations in the stepwise elution. These three scales of chromatography maintained an approximate plasmid yield and the purified plasmid contained undetectable levels of RNA and protein.     

Considerations on the flow configuration of membrane chromatography devices for the purification of human basic fibroblast growth factor from crude lysates

Membrane chromatography possesses numerous advantages such as operation at high flow rates, low back pressure, ease of handling and scale up, which make the membrane adsorber process a viable alternative to conventional packed column chromatography. A purification process for the isolation of human recombinant basic fibroblast growth factor (FGF‐2) based on membrane chromatography was investigated using devices with different flow configurations. In the first process, the FGF‐2 capture step was performed with an axial flow device, while the alternative method achieved direct capture of FGF‐2 from unclarified cell lysate with a tangential flow device. In both processes, FGF‐2 purities exceeded 82% and the purified cytokine displayed high biological activity. Binding capacity (BC) from fermentation broth of the axial flow device was 28 mg/mL. This was 50% higher than the BC obtained with the tangential flow device under particle‐free supernatant conditions (18 mg/mL) and 150% higher compared to the BC achieved with unclarified cell lysate (11 mg/mL). While membrane chromatography in tangential flow mode omits clarification and thus reduces the number of stages in the downstream process, it displays lower peak resolution and leads to a lower overall process yield.     

Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing

Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:974–982, 2015     

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.     

Designing new monoclonal antibody purification processes using mixed-mode chromatography sorbents

Current platforms for purification of monoclonal antibodies, mostly relying on Protein A as a first capture step, are robust and efficient but significantly increase downstream purification costs, mainly due to Protein A resins. To decrease manufacturing costs, industry is increasingly considering the use of purification schemes without affinity Protein A resins. Mixed-mode chromatography can be used as a powerful alternative to standard purification platforms as it offers new selectivity and separation mechanisms exploiting a combination of both ionic and hydrophobic characteristics of antibodies and contaminating proteins. By using a design of experiments (DoE) approach and high throughput screening in 96-well plates, we developed four different two-steps MAb purification processes, based on the use of mixed-mode sorbents. Finally, three of the tested processes resulted in final purified Mab fractions containing less than 100 ppm of residual CHO proteins (CHOP), with overall process yields above 70%. These data show that mixed-mode chromatography sorbents, used at capture or intermediate purification steps, really expand the options of MAb purification process development with or without Protein A affinity chromatography.     

Optimisation of Recombinant Production of Active Human Cardiac SERCA2a ATPase

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca²⁺ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His₈ tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His₈ fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His₈ tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.