Plasmid DNA (pDNA) is a critical starting material for DNA vaccines, messenger RNA (mRNA) production, and gene and cell therapies, and frequently serves either as the final drug substance or as a template for therapeutic molecules. In addition, large plasmid constructs are widely used in transient transfection processes for the production of viral vectors and recombinant proteins, which impose stringent requirements on chromatographic capture and polishing steps. Established bead-based chromatography media, originally developed for protein purification, typically exhibit pore sizes of 30-100 nm, limiting intraparticle mass transfer for pDNA whose size is comparable to or larger than the pore dimensions and resulting in diffusion-limited performance. Consequently, continuous beds or convective flow devices such as monoliths and membrane adsorbers, featuring channels or pores larger than 1 mu m, have emerged as preferred stationary phases for large biomacromolecules, enabling higher productivity at lower pressure drops. This work addresses the scientific question of whether a monolithic structure can be engineered to deliver equal or superior separation performance for supercoiled pDNA-a critical quality attribute-while operating at substantially reduced backpressure. As a model system, pDNA purification was performed using an AXISFLOWTM Q monolith, which is based on an inverted-flow morphology where hollow spheres are packed into a highly interconnected macroporous network with void volumes up to 80%, yielding pressure drops of roughly one third compared with established polymethacrylate monoliths (CIM (R) disks) produced by thermal polymerization. Three plasmids, pUC19 (2.7 kbp), pRep2Cap9 (6.9 kbp), and pHelper (12.3 kbp), were purified and benchmarked against CIM (R) monoliths, cross-linked agarose resins (CaptoTM Q, Q ImpRes), cross-linked copolymer resins (POROSTM 50 HQ, POROSTM XQ), and a membrane adsorber (MustangTM Q XT), focusing on supercoiled yield and product purity. Crude bacterial lysates were processed through two downstream workflows employing either dialysis or tangential flow filtration, and the resulting feed streams were loaded onto the respective anion-exchange devices. Across all conditions and starting materials, the 0.5 mL AXISFLOWTM Q column consistently delivered the highest pDNA yield among the evaluated 1 mL Q-type devices, while meeting GMP-relevant specifications for A260/A280 ratio and supercoiled plasmid content. Levels of process and host-derived impurities, including endotoxin, RNA, and host cell DNA, were at or below required thresholds. These data demonstrate that monolithic materials can be rationally designed to combine low-pressure operation with high-resolution pDNA separation, thereby providing a promising platform for scalable manufacturing of plasmid-based advanced therapies.

Regulatory authorities strongly recommend using residence time distribution (RTD) to achieve material traceability in continuous bioprocesses for non-adsorption units. For adsorption-based units, such as chromatography, retention time distribution (ReTD) is more suitable than RTD for characterizing material flow. Continuous capture chromatography is widely applied for biopharmaceutical continuous manufacturing. However, the ReTD behavior in these systems is still not fully understood. In this study, an ReTD model combining general rate model and two-component mobile phase modulator Langmuir model was developed for Protein A affinity chromatography under high breakthrough conditions. The model was calibrated using adsorption equilibrium experiments, protein breakthrough curves and elution curves. It was then validated through pulse injection experiments at varying protein loading phase. The results showed good agreement between model predictions and experimental results (R2 > 0.945). The exchange mechanism between the solid and liquid phases was further analyzed using confocal laser scanning microscopy images and model simulations, revealing that proteins with stronger binding affinity surpass the bound fraction to bind at the adsorption front while those with weaker affinity would exchange with the surface-bound fractions. Finally, simulations of protein distribution in the column during the interconnected loading step indicate that the exchange effect could broaden the ReTD in continuous chromatography. The model developed lays the groundwork for achieving material traceability and enables non-conforming material diversion strategies to facilitate real-time product release in continuous chromatography processes.

Manufacturing of recombinant proteins in microbial systems, in particular in E. coli, generally requires thorough process development due to the absence of a platform process. The caspase-based fusion process (CASPON (R)) offers a platform manufacturing process using special protease cleavable fusion-tags. These tags allow the implementation of His-tag based affinity chromatography for facile target protein capture and offer expression and solubility enhancing capabilities. The tags are intended as N-terminal fusion motifs that can be fully cleaved using a modified caspase-2 protease, the CASPON (R) enzyme. Here, we systematically explore the influence of various physicochemical parameters on its enzymatic activity. This characterization was performed in parallel in two independent research laboratories using different assays, i.e. a Forster resonance energy transfer-based assay using small peptide substrates and a reversed phase high performance liquid chromatography method using model proteins. Both assays demonstrated great agreement and reveal that CASPON (R) enzyme is highly active at a wide range of temperatures, pH and is resistant to a variety of chemical substances that are commonly employed in bioprocessing, e.g. NaCl, kosmotropes, chaotropes and others. The presence of imidazole at concentrations commonly found in the elution fraction of immobilized metal affinity chromatography does not seem to affect the activity of CASPON (R) enzyme, enabling its use directly after the capture step in downstream processing (DSP). A case study of five biopharmaceuticals is presented. The platform process exhibits consistently high performance in up- and downstream processing, achieving high soluble titers, high yield and purity. The presence of host cell proteins during the DSP in particular was investigated in-depth using process proteomics, revealing that a core of host cell proteins is process dependent and can commonly be expected to be found in the platform DSP. The information presented here can serve as a guide on how to implement the CASPON (R) platform process for the production of various recombinant proteins.

The development of virus-like particle (VLP) production processes is often constrained by the extensive number of analytical methods required for their quantification and characterization, as well as the significant labor demands associated with these techniques. Asymmetrical flow field-flow fractionation (AF4) coupled with in-line detectors, such as ultraviolet (UV) and multi-angle light scattering (MALS), presents a promising label-free and rapid approach to simultaneously assess the quantity and quality of VLP samples. While AF4-MALS has been widely applied for bionanoparticle characterization and quantification in final products and process development, the influence of host cell-derived impurities on the outcome of the analysis remains underexplored. This study investigates the impact of host cell-derived impurities, particularly host cell DNA and chromatin, on AF4-MALS-DLS analysis of both unpurified and purified VLP samples, using HIV-1 gag VLPs produced in CHO cells as a model system. Our results demonstrate that DNA, chromatin, and VLPs can co-elute due to their overlapping size distribution, which, if overlooked, may lead to imprecise determination of VLP concentrations in early process samples and inaccurate yield calculations at later stages. Nevertheless, for total particle quantification, AF4-MALS was shown to be a suitable surrogate for nanoparticle tracking analysis, as the 90° light scattering peak area exhibited a strong linear correlation with total particle concentration. This substitution enables faster sample processing and reduces sample volume requirements. Additionally, our findings highlight the importance of particle concentration and method parameter selection, particularly the detector flow rate, when characterizing samples based on hydrodynamic radius (Rhyd). Underestimation of Rhyd due to high detector flow rates was proposed as the possible explanation for the higher-than-expected shape factors obtained for VLPs. These results emphasize the need for further optimization of AF4 methods to improve the separation of VLPs from host cell impurities and to ensure reliable characterization of bionanoparticles in complex mixtures.

Affinity chromatography-based methods for immunoglobulin G quantification present an attractive alternative to widely used nephelometry due to their simplicity, speed, and compatibility with various sample types. This study validates the efficient analytical use of commercially available POROS CaptureSelect FcXP affinity resin, a stationary phase optimized for fast recombinant human IgG purification. The analytical method was validated in a simple bind-elute mode with a cycle time of 5 minutes. Linearity was confirmed in a range of 4 µg (lower limit of quantification) to 260 µg, allowing direct quantification of IgG from human plasma without further dilution. The method demonstrated excellent precision, with %RSD values ranging from 5% to 1% and satisfactory recovery 99% on average, respectively. By decreasing the residence time from 0.2 minutes to 0.1 minutes, a cycle time of 2.5 minutes was achieved, demonstrating a powerful chromatographic method for fast and reliable IgG quantification. This method offers significant advantages over nephelometry, including reduced analysis time and enhanced compatibility with high-throughput workflows.

Fixed-bed bioreactors for anchorage-dependent cells are an obvious choice for development because of their large-scale capabilities, allowing manufacturing with reduced cost and footprint. In this study, a serum-free production process for Japanese encephalitis virus (JEV) in a single-use fixed-bed bioreactor was developed and compared to conventional roller bottle production as a productivity benchmark. After optimization of serum-free cell culture conditions, an initial media screening in roller bottles showed a strong impact of growth and production media on virus yields. Selected optimized medium combinations were assessed in roller bottles and the fixed-bed bioreactor. Both systems proved to be excellent production systems for JEV, but media choice was key to achieve the highest titers. In particular, DMEM with its enriched glucose content beneficially affected viral yields, enabling potential large-scale manufacturing using the fixed-bed reactor with serum-containing or serum-free media. Practical application: Data presented in this work show feasible ways of serum-free virus production with Vero cells, a common cell substrate in vaccine development. The fixed-bed bioreactor process described here could facilitate manufacturing activities to reduce cost and footprint while simultaneously achieving higher process control compared to conventional manufacturing systems like roller bottles. With a much better upscale potential (up to 500 m2) the fixed-bed bioreactor showed comparable or better yields to roller bottles depending on media used, even with serum-free media. This research article further emphasizes the need to optimize cell culture media or media combinations for each virus individually to achieve the highest titers. As shown, performing a simple media screening experiment to optimize yields early in process development could lead to better productivity, with a high business impact in later development stages.

Downstream processing continues to face significant bottlenecks due to current purification technologies and improvements in upstream. Chromatography systems have been the primary method for purification due to their high yields and purities. However, the use of high-titer-producing strains has highlighted limitations in chromatographic steps, including mass transfer limitations, low capacity, and scalability issues. These challenges, combined with the growing interest in fully continuous manufacturing processes, have led to a widespread interest in alternative to affinity chromatography systems. Polyethylene glycol precipitation has been demonstrated to be a powerful, flexible, easily scalable, and titer-independent methodology for purifying therapeutic proteins such as monoclonal antibodies, achieving yields and purities comparable to chromatography systems. Furthermore, it also holds great potential for simplifying the current purification processes of new modalities and overcome current bottlenecks in downstream processing. Herein, we discuss the latest advances in polyethylene glycol precipitation as a purification technology and explore its future research directions and potential applications.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal microscopy, but also as a tracer for determination of residence time distribution in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. The site occupancy of two labeled antibodies, subclass 1 and 2 conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the IgG1 and IgG2 molecules, in positions 188 and 189, respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling despite the secondary region responsible for binding to the protein A was partly labeled. Fluorescent-labeled antibodies are a good compromise as an inert tracer in protein A affinity chromatography because they are much cheaper than isotope-labeled antibodies.

Secretory immunoglobulin A [sIgA] is a promising candidate for enteric therapeutics applications, and several sIgA-based constructs are currently being developed by groups utilizing clarified Chinese hamster ovary [CHO] cell culture supernatants. To the monoclonal antibody downstream processing typically entails chromatography-based purification processes beginning with Protein A chromatography. In this paper, aqueous two-phase systems [ATPS] were employed for the preliminary purification of secretory immunoglobulin A [sIgA] monoclonal antibody [mAb] from clarified CHO-cell culture supernatants. A 2(4) full factorial design was utilized. The influence of various process parameters such as pH, PEG molecular weight [M-PEG], PEG concentration [C-PEG], and phosphate salt concentration [C-PHO], on the sIgA partition coefficient [K sIgA] and the recovery index [Y] in the PEG phase were evaluated. The Elisa assay revealed that, in the ATPS conditions tested, sIgA mAb was mostly detected in PEG upper phase. Run 14 with the highest sIgA activity exhibited the following conditions: M-PEG 8.000 g/mol, C-PEG 12,5 %, pH 7,0 and C-PHO 10 %, and a sIgA K of 94.50 and a recovery index [Y] of 33.52 %. The proposed platform provides straightforward implementation, yields comparable results, and offers significantly improved economics for manufacturing sIgA mAb biotherapeutics.

Vaccination against influenza virus can reduce the risk of influenza by 40% to 60%, they rely on the production of neutralizing antibodies specific to influenza hemagglutinin (HA) ignoring the neuraminidase (NA) as an important surface target. Vaccination with standardized NA concentration may offer broader and longer-lasting protection against influenza infection. In this regard, we aimed to compare the potency of a NA displayed on the surface of a VLP with a soluble NA. The baculovirus expression system (BEVS) and the novel virus-free Tnms42 insect cell line were used to express N2 NA on gag-based VLPs. To produce VLP immunogens with high levels of purity and concentration, a two-step chromatography purification process combined with ultracentrifugation was used. In a prime/boost vaccination scheme, mice vaccinated with 1 mu g of the N2-VLPs were protected from mortality, while mice receiving the same dose of unadjuvanted NA in soluble form succumbed to the lethal infection. Moreover, NA inhibition assays and NA-ELISAs of pre-boost and pre-challenge sera confirm that the VLP preparation induced higher levels of NA-specific antibodies outperforming the soluble unadjuvanted NA.

The biopharmaceutical industry is rapidly advancing, driven by the need for cutting-edge technologies to meet the growing demand for life-saving treatments. In this context, Model Predictive Control (MPC) has emerged as a promising solution to address the complexity of modern biopharmaceutical production processes. Its ability to optimize operations and ensure consistent product yields has made it an attractive option for manufacturers in this sector. Furthermore, MPC's alignment with the Process Analytical Technology (PAT) initiative provides an additional layer of assurance, facilitating real-time monitoring and enabling swift adjustments to maintain process integrity. This comprehensive review delves into the various applications of MPC, ranging from robust control to stochastic model predictive control, thereby equipping biotechnologists and process engineers with a powerful toolset. By harnessing the capabilities of MPC, as elucidated in this review, manufacturers can confidently navigate the intricate bioprocessing landscape and unlock this approach's full potential in their production processes.

Itaconic acid is a biobased organic acid with clear potential to become a relevant renewable building-block chemical. However, to compete with petrochemical processes, the production cost of biobased processes, particularly the downstream processing contribution, needs to be reduced. In this work, composite membranes comprising ultrathin films of crosslinked poly[(o-cresyl glycidyl ether)-co-formaldehyde] and branched polyethyleneimine on mixed cellulose ester supports were developed for application in the primary recovery of itaconic acid from fermentation broths. In contrast to commercial and literature-reported counterparts, these membranes exhibit low itaconic acid rejections over a wide range of pH, ionic strength, organic acid concentration and operating pressure. Moreover, these membranes can efficiently recover itaconic acid from clarified fermentation broth as well as unclarified fermentation broth of Pichia pastoris cultivations, with average rejections below 15% recorded over 8 hours of operation. Lastly, the performance of these membranes was compared to literature-reported strategies for primary recovery of biobased organic acids. The efficient recovery of itaconic acid from unclarified fermentation broth opens the possibility to perform clarification and primary recovery simultaneously, thus simplifying the downstream processing of biobased itaconic acid considerably.

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.

Advances in affinity chromatography now make it possible to analyze immunoglobulin G from plasma and its fractions with a simple chromatographic method. Ligands derived from camelid antibodies have been developed which have affinity to all 4 subclasses of human IgG without a cross reactivity to other immunoglobulins. The commercially available Capture Select FcXL is the basis for a simple method for direct quantification of immunoglobulin G from plasma or from fractions from cold ethanol precipitation. After direct injection of the sample into the column the unbound proteins are washed out with equilibration buffer and eluted with a pH-step. The elution the peak is integrated, and quantity is derived form a standard curve. The limit of detection with 40 µg/mL, and a linearity up to 250 µg/mL allows an analysis of samples ranging from 0.04 to 50 mg/mL using varying injection volume without further dilution and the two-wavelength detection. A full cycle is completed within five minutes. This method can serve as orthogonal method for in-process control but also for process development.

BACKGROUNDThe determination of the residence time distribution of an entire process or process step in integrated continuous biomanufacturing is required for process understanding, traceability determination and to efficiently handle process disturbances. When recombinant proteins such as monoclonal antibodies are captured by continuous precipitation and two-stage filtration, the question arises whether simple salt is suitable as a tracer or whether a labeled protein is required.RESULTSWe have investigated the use of various inert tracers, sodium nitrate (NaNO3) due to its absorbance at UV 280 nm, and fluorescently labeled or unlabeled antibodies to determine the RTD of the product at steady-state and in the start-up and shut-down phases. All tracers are suitable for measuring the RTD of the precipitated antibody for individual unit operations. For interconnected unit operations, if the product is concentrated, the RTD can be determined with labeled or unlabeled antibodies, because it is not possible to concentrate salts in a microfiltration unit. The duration of the start-up and shut-down phases depends on the concentration factor applied in two-stage filtration, and the number of reactor volumes required to reach steady state corresponds to the concentration factor. Around 1.6 reactor volumes are required for the shutdown phase for 99% wash-out.CONCLUSIONThe integrated process has a plug flow characteristic, which is advantageous as the process can react quickly when process deviations occur. (c) 2024 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Developing an accurate and reliable model for chromatographic separation that meets regulatory requirements and ensures consistency in model development remains challenging. In order to address this challenge, a standardized approach was proposed in this study with ion-exchange chromatography (IEC). The approach includes the following steps: liquid flow identification, system and column-specific parameters determination and validation, multi-component system identification, protein amount validation, steric mass action parameters determination and evaluation, and validation of the calibrated model's generalization ability. The parameter-by-parameter (PbP) calibration method and the consideration of extra-column effects were integrated to enhance the accuracy of the developed models. The experiments designed for implementing the PbP method (five gradient experiments for model calibration and one stepwise experiment for model validation) not only streamline the experimental workload but also ensure the extrapolation abilities of the model. The effectiveness of the standardized approach is successfully validated through an application about the IEC separation of industrial antibody variants, and satisfactory results were observed with R2 ≈ 0.9 for the majority of calibration and validation experiments. The standardized approach proposed in this work contributes significantly to improve the accuracy and reliability of the developed IEC models. Models developed using this standardized approach are ready to be applied to a broader range of industrial separation systems, and are likely find further applications in model-assisted decision-making of process development.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.

Highly purified virus preparations are essential for accurate activity and potency determination. This requires simple and efficient purification methods, especially in the early stages of research and development. While heparin affinity chromatography has been already successfully used for the purification of several enveloped viruses and virus-like particles, we extended its use to purification of very sensitive measles virus. The performance of heparin and heparin-like affinity chromatography was evaluated for the purification of recombinant measles virus, a large and labile enveloped virus used as vaccine or cancer therapy. Since DNA, particularly in the form of chromatin is a critical impurity in enveloped virus preparations, the effect of integration of an endonuclease (Benzonase® or M-SAN) treatment prior to chromatography was also investigated. Both, Capto™ DeVirS (heparin-like) and Capto™ Heparin were able to capture measles viruses directly from clarified cell culture supernatant. Despite capturing 100 % of infectious measles virus, low recovery (8 %) was observed for Capto™ DeVirS. For Capto™ Heparin recoveries up to 85 % were observed. The combination of M-SAN with Capto™ Heparin enabled the production of highly purified measles virus with a yield of 62 % and a final purity of 10.2 ng dsDNA per dose (1 × 105), outperforming the processes without endonuclease treatment with a yield of 18 %, and a purity of 66.7 ng dsDNA/dose or using Benzonase® with a yield of 38 % and a purity of 21.2 ng dsDNA/dose. As the developed method is simple and scalable it could also be integrated in a downstream process train for measles virus manufacturing.

Chimeric virus-like particles formed by the co-expression of HIV-1 gag capsid protein and surface proteins from other viruses can be used as a platform for production of a wide range of viral vaccines. These virus-like particles can be rapidly produced in an insect cell culture using the baculovirus expression vector system. However, a variety of different process-related impurities such as host cell proteins, double stranded DNA, chromatin, and structurally similar bionanoparticles like extracellular vesicles and baculovirus particles are present in the harvested supernatant and complicate the chromatographic purification steps. Heparan sulfate is the first entry point of a lot of enveloped viruses into the cell. Taking advantage of this functional property, its sub-variant, the glycosaminoglycan heparin immobilized on a chromatography media is a universal tool for the separation of enveloped virus-like particles. A purification process was developed consisting of clarification of the cell culture supernatant by membrane filtration, a pre-purification step using core-shell bead flow-through chromatography with CaptoTM Core 700, followed by heparin affinity chromatography with CaptoTM Heparin. The combination of CaptoTM Core 700 and CaptoTM Heparin allows for direct load of the flow-through material from the first into the second step without buffer exchange in between. In addition, lengthy buffer screening, as required for ion exchange and multimodal chromatography, can be omitted. Linear salt gradient elution in heparin affinity chromatography enabled separation of virus-like particles and baculovirus. The platformability of the process was demonstrated by purification of virus-like particles expressing influenza A virus hemagglutinin or neuraminidase, or SARS-CoV-2 spike protein as surface antigens and the virus-like particle without co-expressed surface protein. Western blotting, host cell protein and DNA assays, TCID50 (for residual baculovirus), mass spectrometry, multi-angle light scattering and nanoparticle tracking analysis were used to characterize the different fractions obtained during the purification. A similar elution profile was obtained for all tested virus-like particles and deviations could be explained by batch-to-batch variations and differences in productivity for the different constructs. Double stranded DNA was reduced to 7 - 36 ng/109 particles and host cell protein content was below the limit of quantification; in the main product fraction an average seven-fold reduction of baculovirus and a yield of 9 % - 14 % of bound particles with a concentration of 1.4 - 1.8 x 1010 particles/mL in the eluate were achieved meeting requirements for a viral vaccine. Accordingly, this process is suitable as a downstream process platform for chimeric HIV-1 gag-based VLP products.