Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants.

This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography-mass spectrometry (LC-MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M255, W420, and M431) was observed for peptides situated at the Fc region close to the CH2-CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.

Residence time distribution in continuous virus filtration.

Regulatory authorities recommend using residence time distribution (RTD) to address material traceability in continuous manufacturing. Continuous virus filtration is an essential but poorly understood step in biologics manufacturing in respect to fluid dynamics and scale-up. Here we describe a model that considers nonideal mixing and film resistance for RTD prediction in continuous virus filtration, and its experimental validation using the inert tracer NaNO3. The model was successfully calibrated through pulse injection experiments, yielding good agreement between model prediction and experiment ( R 2 > ${R}^{2}\gt $ 0.90). The model enabled the prediction of RTD with variations-for example, in injection volumes, flow rates, tracer concentrations, and filter surface areas-and was validated using stepwise experiments and combined stepwise and pulse injection experiments. All validation experiments achieved R 2 > ${R}^{2}\gt $ 0.97. Notably, if the process includes a porous material-such as a porous chromatography material, ultrafilter, or virus filter-it must be considered whether the molecule size affects the RTD, as tracers with different sizes may penetrate the pore space differently. Calibration of the model with NaNO3 enabled extrapolation to RTD of recombinant antibodies, which will promote significant savings in antibody consumption. This RTD model is ready for further application in end-to-end integrated continuous downstream processes, such as addressing material traceability during continuous virus filtration processes.

Status and future developments for downstream processing of biological products: Perspectives from the Recovery XIX yield roundtable discussions.

Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models, and global strategies to make a COVID-19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low- and middle-income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody-drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics-like cell and gene therapies. The industry must develop a model-based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work-life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.

Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation.

Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ion-exchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C. Isolated charge variants arising under stress conditions were characterized by peptide mapping. The results of peptide mapping showed that deamidation in the Fc domain and N-terminal pyroglutamate formation in the heavy chain are the main contributors to charge heterogeneity. The heavy chain CDR2, which is the only CDR containing asparagine residues, was quite resistant to deamidation under stress conditions according to peptide mapping results. Using surface plasmon resonance, it was shown that the affinity of pertuzumab for the HER2 target receptor does not change under stress conditions. Peptide mapping analysis of clinical samples showed an average of 2-3% deamidation in the heavy chain CDR2, 20-25% deamidation in the Fc domain, and 10-15% N-terminal pyroglutamate formation in the heavy chain. These findings suggest that in vitro stress studies are able to predict in vivo modifications.

Sensors and chemometrics in downstream processing.

The biopharmaceutical industry is still running in batch mode, mostly because it is highly regulated. In the past, sensors were not readily available and in-process control was mainly executed offline. The most important product parameters are quantity, purity, and potency, in addition to adventitious agents and bioburden. New concepts using disposable single-use technologies and integrated bioprocessing for manufacturing will dominate the future of bioprocessing. To ensure the quality of pharmaceuticals, initiatives such as Process Analytical Technologies, Quality by Design, and Continuous Integrated Manufacturing have been established. The aim is that these initiatives, together with technology development, will pave the way for process automation and autonomous bioprocessing without any human intervention. Then, real-time release would be realized, leading to a highly predictive and robust biomanufacturing system. The steps toward such automated and autonomous bioprocessing are reviewed in the context of monitoring and control. It is possible to integrate real-time monitoring gradually, and it should be considered from a soft sensor perspective. This concept has already been successfully implemented in other industries and requires relatively simple model training and the use of established statistical tools, such as multivariate statistics or neural networks. This review describes a scenario for integrating soft sensors and predictive chemometrics into modern process control. This is exemplified by selective downstream processing steps, such as chromatography and membrane filtration, the most common unit operations for separation of biopharmaceuticals.

Increase in cysteine-mediated multimerization under attractive protein-protein interactions.

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein-protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein-protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.

PROFICS: A bacterial selection system for directed evolution of proteases.

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

Characterization of hydrodynamics and volumetric power input in microtiter plates for the scale-up of downstream operations.

Parameter estimation for scale-up of downstream operations from microtiter plates (MTPs) is mostly done empirically because engineering correlations between microplates and stirred tank reactors (STRs) are not yet available. It is challenging to change the operation mode from shaken MTPs to large-scale STRs. For the scale-up of STRs, volumetric power input is well-established although it is unclear whether this parameter can be used to transfer the operations from MTPs. We determine the volumetric power input in MTPs via the temperature increase caused by the motion of the liquid. The hydrodynamics in MTPs are studied with computational fluid dynamics (CFD). Mixing is investigated in 96-, 24-, and 6-well MTPs to cover different geometries, filling volumes, shaking diameters, and shaking frequencies. All CFD simulations are validated by experimental results, which now allows prediction of the volumetric power input and hydrodynamics at various conditions in MTPs without the need for further experiments. We provide a map of the power input achievable in MTPs. Based on this map, from knowing about large-scale conditions, adequate microscale conditions can be adjusted for process development. This enables the direct scale-up of downstream unit operations from MTPs to STRs.

Prediction of the performance of pre-packed purification columns through machine learning.

Pre-packed columns have been increasingly used in process development and biomanufacturing thanks to their ease of use and consistency. Traditionally, packing quality is predicted through rate models, which require extensive calibration efforts through independent experiments to determine relevant mass transfer and kinetic rate constants. Here we propose machine learning as a complementary predictive tool for column performance. A machine learning algorithm, extreme gradient boosting, was applied to a large data set of packing quality (plate height and asymmetry) for pre-packed columns as a function of quantitative parameters (column length, column diameter, and particle size) and qualitative attributes (backbone and functional mode). The machine learning model offered excellent predictive capabilities for the plate height and the asymmetry (90 and 93%, respectively), with packing quality strongly influenced by backbone (∼70% relative importance) and functional mode (∼15% relative importance), well above all other quantitative column parameters. The results highlight the ability of machine learning to provide reliable predictions of column performance from simple, generic parameters, including strategic qualitative parameters such as backbone and functionality, usually excluded from quantitative considerations. Our results will guide further efforts in column optimization, for example, by focusing on improvements of backbone and functional mode to obtain optimized packings.

Model predictive control for steady-state performance in integrated continuous bioprocesses.

Perfusion bioreactors are commonly used for the continuous production of monoclonal antibodies (mAb). One potential benefit of continuous bioprocessing is the ability to operate under steady-state conditions for an extended process time. However, the process performance is often limited by the feedback control of feed, harvest, and bleed flow rates. If the future behavior of a bioprocess can be adequately described, predictive control can reduce set point deviations and thereby maximize process stability. In this study, we investigated the predictive control of biomass in a perfusion bioreactor integrated to a non-chromatographic capture step, in a series of Monte-Carlo simulations. A simple algorithm was developed to estimate the current and predict the future viable cell concentrations (VCC) of the bioprocess. This feature enabled the single prediction controller (SPC) to compensate for process variations that would normally be transported to adjacent units in integrated continuous bioprocesses (ICB). Use of this SPC strategy significantly reduced biomass, product concentration, and harvest flow variability and stabilized the operation over long periods of time compared to simulations using feedback control strategies. Additionally, we demonstrated the possibility of maximizing product yields simply by adjusting perfusion control strategies. This method could be used to prevent savings in total product losses of 4.5-10% over 30 days of protein production.

Fusion Tag Design Influences Soluble Recombinant Protein Production in Escherichia coli.

Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.

Media on-demand: Continuous reconstitution of a chemically defined media directly from solids.

Chemically defined media are reconstituted batchwise and stored in hold tanks until use. To avoid large hold tanks and batchwise production of media, we developed continuous on-demand reconstitutions directly from solids consisting of a hopper and a screw conveyor capable of feeding dry powdered media with the required precision ±5% at low dosing rates of 0.171 g min-1 . A commercially available dry powdered cell culture medium was continuously fed over a duration of 12 h into a mixer which was connected to a UV-cell for monitoring and the media were compared to a batchwise production. A comparable amino acid, carbohydrate, and osmolality profile to a batchwise reconstitution could be obtained. Cell cultivation showed comparable performance of batch and continuous reconstitution for two CHO cell lines producing the antibodies adalimumab and trastuzumab on a small and benchtop scale. In-depth analysis of the produced antibodies showed the same glycosylation pattern, other posttranslational profiles such as methionine oxidation and deamidation compared to batchwise reconstitution. Therefore, we conclude a continuous reconstitution of the medium results in the same quality of the product. A continuous on-demand media reconstitution will impact the supply chain and significantly reduce the floor space necessary for preparation and storage.

Technology transfer of a monitoring system to predict product concentration and purity of biopharmaceuticals in real-time during chromatographic separation.

Technological developments require the transfer to their location of application to make use of them. We describe the transfer of a real-time monitoring system for lab-scale preparative chromatography to two new sites where it will be used and developed further. Equivalent equipment was used. The capture of a biopharmaceutical model protein, human fibroblast growth factor 2 (FGF-2) was used to evaluate the system transfer. Predictive models for five quality attributes based on partial least squares regression were transferred. Six out of seven online sensors (UV/VIS, pH, conductivity, IR, RI, and MALS) showed comparable signals between the sites while one sensor (fluorescence) showed different signal profiles. A direct transfer of the models for real-time monitoring was not possible, mainly due to differences in sensor signals. Adaptation of the models was necessary. Then, among five prediction models, the prediction errors of the test run at the new sites were on average twice as high as at the training site (model-wise 0.9-5.7 times). Additionally, new prediction models for different products were trained at each new site. These allowed monitoring the critical quality attributes of two new biopharmaceutical products during their purification processes with mean relative deviations between 1% and 33%.

Truly continuous low pH viral inactivation for biopharmaceutical process integration.

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities-which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

Separation of influenza virus-like particles from baculovirus by polymer-grafted anion exchanger.

The baculovirus expression vector system is a very powerful tool to produce virus-like particles and gene-therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for wider industrial use. We used chimeric human immunodeficiency virus-1 (HIV-1) gag influenza-hemagglutin virus-like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus-like particles. A fast and simple purification method for these virus-like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer-grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus-like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography-mass spectrometry. When considering a vaccination dose of 109 particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double-stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus-like particles.

Continuous integrated antibody precipitation with two-stage tangential flow microfiltration enables constant mass flow.

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two-stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two-stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre-existing high-molecular-weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter-current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow-through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface.

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody-protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.

Real-time monitoring and model-based prediction of purity and quantity during a chromatographic capture of fibroblast growth factor 2.

Process analytical technology combines understanding and control of the process with real-time monitoring of critical quality and performance attributes. The goal is to ensure the quality of the final product. Currently, chromatographic processes in biopharmaceutical production are predominantly monitored with UV/Vis absorbance and a direct correlation with purity and quantity is limited. In this study, a chromatographic workstation was equipped with additional online sensors, such as multi-angle light scattering, refractive index, attenuated total reflection Fourier-transform infrared, and fluorescence spectroscopy. Models to predict quantity, host cell proteins (HCP), and double-stranded DNA (dsDNA) content simultaneously were developed and exemplified by a cation exchange capture step for fibroblast growth factor 2 expressed in Escherichia coliOnline data and corresponding offline data for product quantity and co-eluting impurities, such as dsDNA and HCP, were analyzed using boosted structured additive regression. Different sensor combinations were used to achieve the best prediction performance for each quality attribute. Quantity can be adequately predicted by applying a small predictor set of the typical chromatographic workstation sensor signals with a test error of 0.85 mg/ml (range in training data: 0.1-28 mg/ml). For HCP and dsDNA additional fluorescence and/or attenuated total reflection Fourier-transform infrared spectral information was important to achieve prediction errors of 200 (2-6579 ppm) and 340 ppm (8-3773 ppm), respectively.

A two-step process for capture and purification of human basic fibroblast growth factor from E. coli homogenate: Yield versus endotoxin clearance.

A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/µg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 µg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/µg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV280nm or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria.

At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification.

At-line static light scattering and fluorescence monitoring allows direct in-process tracking of fluorescent virus-like particles. We have demonstrated this by coupling at-line multi-angle light scattering and fluorescence detectors to the downstream processing of enveloped virus-like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus-like particles containing the Human Immunodeficiency Virus-1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single-column anion-exchange chromatography method was used for direct capture and purification. The majority of host-cell protein impurities passed through the column without binding. Virus-like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi-angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus-like particles were concentrated 17-fold to final a concentration of 4.45 × 1010 particles/mL. Simple buffers and operation make this process suitable for large scale purposes.