Chromatographic Media Research Articles

Macroporous cellulose microspheres with high specific surface area via semi-dissolved chitosan templating for protein separation

The conflict between macroporous structure and high specific surface area strongly limited the dynamic adsorption performance of chromatographic media. Here, macroporous and high-specific-surface-area cellulose microspheres (PCMs) are prepared by using semi-dissolved chitosan as a dual-functional template and subsequently functionalized with diethylaminoethyl (DEAE) as anion exchangers for protein separation. The dual-functional template strategy leverages dissolved chitosan nanofibers as meso/micropore templates and the undissolved chitosan particles as macropore templates. The macropores of DEAE-PCMs (average size: 0.4–1.2 μm) efficiently decrease mass transfer resistance for fast uptake kinetics. The high specific surface area (272.34 m2/g) provides sufficient ligand sites for ensuring exceptional adsorption capacities. Therefore, the chitosan bifunctional templated PCMs, possessing a macro/meso/microporous structure, are promising candidates for high-performance chromatographic media.

Screening and isolation of quorum sensing inhibitors of Pseudomonas aeruginosa from Phellodendron amurense extracts using bio-affinity chromatography.

Drug-resistant bacterial infections pose a significant challenge in the field of bacterial disease treatment. Finding new antibacterial pathways and targets to combat drug-resistant bacteria is crucial. The bacterial quorum sensing (QS) system regulates the expression of bacterial virulence factors. Inhibiting bacterial QS and reducing bacterial virulence can achieve antibacterial therapeutic effects, making QS inhibition an effective strategy to control bacterial pathogenicity. This article mainly focused on the PqsA protein in the QS system of Pseudomonas aeruginosa. An affinity chromatography medium was developed using the SpyTag/SpyCatcher heteropeptide bond system. Berberine, which can interact with the PqsA target, was screened from Phellodendron amurense by affinity chromatography. We characterized its structure, verified its inhibitory activity on P. aeruginosa, and preliminarily analyzed its mechanism using molecular docking technology. This method can also be widely applied to the immobilization of various protein targets and the effective screening of active substances.

Regulation of macroporous cellulose microspheres via phase separation force induced by carbon nanotubes doping for enhanced protein adsorption

The burgeoning requirement for purified biomacromolecules in biopharmaceutical industry has amplified the exigency for advanced chromatographic separation techniques. Herein, macroporous cellulose microspheres (CCMs) with micron-sized pores are produced by a facile regulation via carbon nanotubes (CNTs). In this strategy, the incorporation of CNTs breaks the homogeneous regeneration of the cellulose, thus providing anisotropic phase force to produce macropores. The CCMs have manifested a faster mass transfer rate and more available adsorption sites owing to well-defined macropores (2.69 ± 0.57 μm) and high specific surface area (147.47 m2 g−1). Further, CCMs are functionalized by quaternary ammonium salts (GTAc-CCMs) and utilized as anion adsorbents to adsorb pancreatic kininogenase (PK). The prepared GTAc-CCMs show rapid adsorption kinetics for PK at pH 6.0, reaching 90 % equilibrium within 60 min. Also, GTAc-CCMs for PK exhibit high adsorptive capacity (632.50 mg g−1), excellent recyclability (> 80 % removal amount after 10 cycles) and selectivity especially at pH 6.0. Notably, the GTAc-CCMs have been successfully applied in a fixed-bed chromatography process, indicating their potential as an effective chromatographic medium for rapid separation of biomacromolecules.

Purification of Adeno-Associated Viral Vector Serotype 9 Using Ceramic Hydroxyapatite Chromatography and its Analysis.

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.

Competitive sorption of low and high molecular weight proteins in model mixtures by Q HyperZ chromatographic medium. Part II: Mass transfer mechanisms

The kinetic uptake of Bovine Serum Albumin (BSA) and ferritin in mixtures by the composite anion exchanger Q HyperZ was studied. Transient adsorption experiments were performed to investigate the dominant resistance between external and internal mass transfer in simultaneous and sequential adsorption experiments. From the evolution of protein solution concentration with time, the external mass transfer coefficients determined were very low in simultaneous (7.1 10−7 and 1.1 10−7 m/s for BSA and ferritin respectively) and sequential (4.6 10−7 and 2.5 10−7 m/s for BSA and ferritin respectively) adsorption systems which is in agreement with the low agitation speed used. From the transient protein uptakes, the internal mass transfer was studied and the homogenous diffusion model applied provides a good predictability in single as well as in simultaneous and sequential adsorption experiments. The effective diffusion coefficients were determined in sequential (6.4 10−14 m2/s and 3.8 10−14 m2/s for BSA and ferritin respectively) and simultaneous (4.2 10−14 m2/s for both proteins) systems. In multi-component adsorption system, it was found that BSA was slowed down upon the presence of ferritin on mixture solutions contrarily to ferritin protein. This phenomenon, known as electrostatic coupling effect, indicates that diffusion of charged proteins in ion exchangers are not independent.

Preparation of a macroporous anion exchange chromatographic medium by polyamine grafting and evaluation of its protein-adsorption behavior

The macroporous anion exchange chromatographic medium (FastSep-PAA) was prepared through grafting polyallylamine (PAA) onto polyacrylate macroporous microspheres (FastSep-epoxy). The effects of the synthesis conditions, including the PAA concentration, reaction time, and reaction solution pH, on the ion exchange (IC) of the medium were investigated in detail. When the PAA concentration, reaction time, and reaction solution pH were increased, the IC of the medium increased, and optimal synthesis conditions were then selected in combination with changes of protein binding capacity. A scanning electron microscope was used to examine the surface morphology of the medium. The medium possessed high pore connectivity. Furthermore, the pore structure of the medium was preserved after the grafting of PAA onto the macroporous microspheres. This finding demonstrates that the density of the PAA ligands does not appear to have any discernible impact on the structure of the medium; that is, no difference in the structure of the medium is observed before and after the grafting of PAA onto the microspheres. The pore size and pore-size distribution of the medium before and after grafting were determined by mercury intrusion porosimetry and the nitrogen adsorption method to investigate the relationship between pore size (measured in the range of 300-1000 nm) and protein adsorption. When the pore size of the medium was increased, its protein binding capacity did not exhibit any substantial decrease. An increase in pore size may hasten the mass transfer of proteins within the medium. Among the media prepared, that with a pore size of 400 nm exhibited the highest dynamic-binding capacity (DBC: 70.3 g/L at 126 cm/h). The large specific surface area of the medium and its increased number of protein adsorption sites appeared to positively influence its DBC. When the flow rate was increased, the protein DBC decreased in media with original pore sizes of less than 700 nm. In the case of the medium with an original pore size of 1000 nm, the protein DBC was independent of the flow rate. The protein DBC decreased by 3.5% when the flow rate was increased from 126 to 628 cm/h. In addition, the protein DBC was maintained at 57.7 g/L even when the flow velocity was 628 cm/h. This finding reveals that the diffusion rate of protein molecules at this pore size is less restricted and that the prepared medium has excellent mass-transfer performance. These results confirm that the macroporous polymer anion exchange chromatographic medium developed in this study has great potential for the high-throughput separation of proteins.

Capture of fully assembled secretory immunoglobulin A by affinity chromatography with nanobodies as ligands

Secretory immunoglobulin A (sIgA) has strongpotential for passive immunization and other therapeutic applications. This complex molecule with a molecular mass of 410 kDa consists of two IgA monomers linked by a connecting chain (J) and a secretory component (SC). Fully assembled sIgA was overexpressed in CHO cells. In order to exploit the therapeutic potential of recombinant sIgA expressed in CHO cells, a platform process for capture of the antibody directly from the clarified culture supernatant using available affinity chromatography material was investigated. The hydrodynamic radius was 10.2 ± 0.9 nm determined by dynamic light scattering and the isoelectric point 5.5 by electrophoretic mobility using the Malvern Zetasizer. Capture Select IgA and Capture Select IgA-CH1 chromatography media with camelid-derived single-domain antibody fragments comprising the 3 CDR domain antibody fragment with a 13 kD or 14 kDa as ligand were used to purify the antibody. sIgA was directly captured from the clarified culture supernatant and a highly purified, full-length, correctly assembled sIgA was obtained. The residence time must be in the range of 5–10 min to achieve a high binding capacity. The effective diffusivity of sIgA was in the range of 10−9 cm2 s−1 and the dynamic binding capacity was in the range of 10 mg/ml packed bed. We conclude that the current version of the resin has a pore size that is too small for this type of molecule to achieve substantial resin utilization and productivity, although affinity ligand-based purification led to high degree of purity suited for an industrial capture process.

Improved mRNA affinity chromatography binding capacity and throughput using an oligo-dT immobilized electrospun polymer nanofiber adsorbent

Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3–0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900–4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2–4-fold higher than CIMmultus® and ∼2–13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media.

Evaluation of the Robustness Verification of Downstream Production Process for Inactivated SARS-CoV-2 Vaccine and Different Chromatography Medium Purification Effects.

Large-scale vaccine production requires downstream processing that focuses on robustness, efficiency, and cost-effectiveness. To assess the robustness of the current vaccine production process, three batches of COVID-19 Omicron BA.1 strain hydrolytic concentrated solutions were selected. Four gel filtration chromatography media (Chromstar 6FF, Singarose FF, Bestarose 6B, and Focurose 6FF) and four ion exchange chromatography media (Maxtar Q, Q Singarose, Diamond Q, and Q Focurose) were used to evaluate their impact on vaccine purification. The quality of the vaccine was assessed by analyzing total protein content, antigen content, residual Vero cell DNA, residual Vero cell protein, and residual bovine serum albumin (BSA). Antigen recovery rate and specific activity were also calculated. Statistical analysis was conducted to evaluate process robustness and the purification effects of the chromatography media. The statistical analysis revealed no significant differences in antigen recovery (p = 0.10), Vero HCP residue (p = 0.59), Vero DNA residue (p = 0.28), and BSA residue (p = 0.97) among the three batches of hydrolytic concentrated solutions processed according to the current method. However, a significant difference (p < 0.001) was observed in antigen content. The study demonstrated the remarkable robustness of the current downstream process for producing WIBP-CorV vaccines. This process can adapt to different batches of hydrolytic concentrated solutions and various chromatography media. The research is crucial for the production of inactivated SARS-CoV-2 vaccines and provides a potential template for purifying other viruses.

Model‐based optimization of an ion exchange chromatography process for the separation of von Willebrand factor fragments and human serum albumin

AbstractColumn liquid chromatography plays a vital role in the downstream processing of biopharmaceuticals, where the goal is to extract and purify a target protein from a mixture. In this work, we examine a real‐world ion exchange chromatography process where electrostatic interactions between proteins and the chromatographic medium are the predominant forces being exploited for the separation of the participating species. More specifically, we successfully separate a recombinant dimeric fragment of the von Willebrand factor, used for the half‐life prolongation of certain proteins, from human serum albumin. Compared to the originally conducted experiments, the optimized experiment achieves a higher product purity in less time and with lower consumption of buffer salts, hence being favorable both in economical and ecological terms.

Nonadditivity and Nonlinearity of Mobile and Stationary Zone Mass Transfer Resistances in Chromatography.

Using a two-zone moment analysis (TZMA) method based on Brenner's generalized dispersion theory for two-dimensional (2D) and three-dimensional (3D) periodic media, we investigated the mechanisms for dispersion in particulate media for liquid chromatography. This was done using a set of plate height data covering an unprecedented wide range of retention factors, diffusion coefficients, and velocities, all computed with unequaled accuracy. Applying Giddings' additivity test, based on alternatingly making the diffusion coefficient in the mobile and stationary zones infinitely large, the dispersion data clearly indicate a lack of additivity. Although this lack could be directly understood by identifying the existence of multiple parallel mass transfer paths, the additivity assumption interestingly overestimates the true C term band broadening (typically by more than 10%, depending on conditions and dimensionality of the system). However, Giddings originally asserted the occurrence of parallel paths would always lead to an underestimation of the dispersion. The origin of the lack of additivity is analyzed in detail and qualitatively explained. Finally, we also established a generic framework for the modeling of the effect of the reduced velocity and the retention coefficient on the C term in ordered chromatographic media. This led to the introduction of a new expression for the mobile zone mass transfer term, which, unlike the currently used literature expression, contains the complete k″ dependency.

High Recovery Chromatographic Purification of mRNA at Room Temperature and Neutral pH.

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5-7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3-7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature.

Eco-friendly and underwater superelastic cellulose nanofibrous aerogels for efficient capture and high-throughput protein separation

High-performance and environmentally compatible chromatographic media with superb adsorption capacity and large processing throughput is key to efficient protein separation. Herein, composite aerogels (CCS-NFAs) of cellulose nanofibrous and chitosan (CCS) with 3D lamellar structure were developed by freeze-drying method for adsorptive separation of protein. The CCS-NFAs exhibited good under water strength (25.2 kPa at 60 % compression strain) and underwater superelasticity despite being highly porous (over 98%) and lightweight (10.94 mg cm−3). The maximum adsorption capacity for bovine serum albumin (BSA) was calculated to be about 1269 mg g−1, and the corresponding dynamic adsorption capability was as high as 914 mg g−1 with a filling mass of 20 mg at a flow rate of 30 mL h−1, compared favorably against the state-of-the-art ion-exchange chromatography materials. The superb adsorptive capacity was due to the involvement of chemical adsorption as confirmed by the pseudo-second-order kinetic model and Langmuir model fitting of the experimental data. Additionally, the highly oriented large open-cells in the material enabled ultrahigh water flux at about 2.2 × 104 L m-2h−1, which showed no significant attenuation in flux or removal efficiency. The successful preparation of CCS-NFAs contributes to protein purification engineering by offering an eco-friendly high-performance chromatographic media.

Bacterial cellulose-based submicrobundle framework-induced hyperporous cellulose microspheres functionalized by ionic liquids for anion exchange chromatography

Ovalbumin (OVA) is widely used in food and pharmaceutical industries and can be separated using ion exchange chromatography. However, both rapid adsorption kinetics and great adsorption capacity are impeded by the nanoscale porous structures of chromatography media. Here, hyperporous cellulose microspheres (BCMs) with well-controllable micron-sized macropores are fabricated by a facile one-step pre-crosslinking strategy. Bacterial cellulose nanofibers and dissolved cellulose chains are pre-crosslinked by epichlorohydrin to form submicrobundles and generate an interconnected gel network. The preliminary gelation blocks induce a hyperporous framework during the recrystallization and assembly of regenerated cellulose. BCMs have demonstrated decreased mass transfer resistance and increased accessible active sites due to the micron-sized convective macropores (2.04 μm) and high specific surface area (194.25 m2 g−1). Cationic imidazolium ILs (AMIBr) functionalized BCMs (IL-BCMs) are prepared as anion exchangers. IL-BCMs exhibit rapid adsorption kinetics for OVA, reaching 85% of the equilibrium adsorption amount within 30 min. Additionally, IL-BCMs demonstrate endothermic chemosorption, high adsorption capacity (150.30 mg g−1, 25 °C), reusability (>85% removal efficiency after seven cycles), and selectivity towards OVA at pH 6.0. Continuous fixed-bed adsorption further suggests the practicality of IL-BCMs in ion exchange chromatography, indicating that IL-BCMs are appealing media for protein separation and purification.

Application of cellulose to chromatographic media: Cellulose dissolution, and media fabrication and derivatization

As the cornerstone of chromatographic technology, the development of high-performance chromatographic media is a crucial means to enhance the purification efficiency of biological macromolecules. Cellulose is a popular biological separation medium due to its abundant hydroxyl group on the surface, easy modification and, weak non-specific adsorption. In this paper, the development of cellulosic solvent systems, typical preparation methods of cellulosic chromatographic media, and the enhancement of chromatographic properties of cellulosic chromatographic media by polymeric ligand grafting strategies and their mechanism of action are reviewed. Ultimately, based on the current research status, a promising outlook for the preparation of high-performance cellulose-based chromatographic media was presented.

Deconvolution of multichannel LC-MS/MS chromatograms of glucosamine-phosphates: Evidence of a GlmS regulatory difference between Staphylococcus aureus and Enterococcus faecium

Resolving isomeric analytes is challenging given their physical similarity – making chromatographic resolution difficult, and their identical masses – making simple mass resolution impossible. MS/MS data provides a means to resolve isomeric analytes if their MS/MS intensity profiles are sufficiently different. Glucosamine-6-phosphate (GlcN-6P) and glucosamine-1-phosphate (GlcN-1P) are early bacterial cell wall intermediates. These and other isomeric hexosamine-phosphates are highly polar and unretained on reverse-phase chromatography media. Three commercially available hexosamine-phosphate standards (GlcN-6P, GlcN-1P, and GalN-1P) were derivatized with octanoic anhydride, and chromatographic conditions were established to resolve these analytes on C18 columns. GlcN-1P and GalN-1P overlapped chromatographically under all tested chromatography conditions. Three MS/MS fragments (79, 97, and 199 m/z) were common to all three commercially available hexosamine-phosphates. Intensity ratios of the three MS/MS fragments from these three hexosamine-phosphate standards were used to deconvolute mixture chromatograms of these standards by non-negative linear regression. This approach allowed the complete resolution of these analytes. The chromatographically overlapping GlcN-1P and GalN-1P, which shared similar but modestly different MS/MS intensity profiles, were fully resolved with this non-negative deconvolution approach. This approach was then applied to MRSA, VSE, and VRE bacterial extracts before and after exposure to vancomycin. This demonstrated a substantial (3-fold) increase in GlcN-6P in vancomycin-treated MRSA samples but not in vancomycin-treated VSE or VRE samples. These observations appear to localize previously observed differences between MRSA and VRE/VSE peptidoglycan biosynthesis regulation to GlmS, which synthesizes GlcN-6P and is the product of a regulatory ribozyme sensitive to the levels of GlcN-6P

Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents.

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014TP mL-1 (RT = 1.6s), with a pressure drop variation of 0.14MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.

Efficient adsorption of alginate oligosaccharides by ion exchange resin based on molecular simulation and experiments

Alginate oligosaccharides (AOS) with low polymerization have gained considerable attention due to their unique physiological activities. However, the lack of understanding regarding the mechanism of action for resin selection and optimization of separation processes has impeded their precise chromatographic separation and large-scale production. Here, the adsorption–desorption characteristics of AOS on the resins with varying solid phase composition, including backbones, functional groups, crosslinking degrees, and counterions resin structures, were analyzed using simulations and experiments. The simulation results revealed that the structural differences of AOS are characterized by their electrostatic potential and lowest unoccupied molecular orbital energy level. Based on molecular simulations, we predicted that the basic anion resin with a styrene backbone would be suitable for AOS separation. Subsequently, we constructed SM-7Cl resin based on the simulation results and performed static adsorption experiments to verify the prediction. SM-7Cl resin demonstrated noteworthy adsorption–desorption performance for AOS, with an adsorption capacity of 471.2 mg/g and a desorption rate of 89.45%. The fitting adsorption isotherm was consistent with the Freundlich model, indicating that the adsorption proceeded spontaneously. The equilibrium times for AOS adsorption on the gel-type resin and the macroporous resin, which both followed a pseudo-second-order kinetic model, were 30 min and 60 min, respectively. This study provides optimized chromatographic media and separation processing that can be implemented for the amplification of chromatographic separation in AOS production.

Conjugated surfactant micelles: A non‐denaturing purification platform for concentrated human immunoglobulin G

AbstractAs downstream purification and separation technologies progress towards raising the concentration of therapeutic‐grade monoclonal antibodies (mAbs) in cell culture, downstream processing has begun to face increased difficulty in efficiently coping with such high immunoglobulin G (IgG) titers (≤25 mg mL−1). In the current study, we demonstrate the ability of a non‐chromatographic, ligand‐free procedure to recover almost quantitatively (84–99% yield, by densitometry) polyclonal, human IgG present at high concentrations (15–25 mg mL−1) in E. coli lysate. Instead of chromatographic media and columns, we use conjugated, mixed‐micelles comprising non‐ionic detergents, tyrosine monomers, and the amphiphilic [(bathophenanthroline)3:Fe2+] complex. Capture and extraction processes are performed at pH 6.5–7, thereby avoiding antibody exposure to acidic, potentially denaturing conditions. Recovered IgG is monomeric as determined by dynamic light scattering (DLS). Process upscaling from 0.1 to 5 mL requires only proportional increase in all reagents and does not affect overall yield or antibody purity.

Removal of chromatin by salt-tolerant endonucleases for production of recombinant measles virus.

Host cell DNA is a critical impurity in downstream processing of enveloped viruses. Especially, DNA in the form of chromatin is often neglected. Endonuclease treatment is an almost mandatory step in manufacturing of viral vaccines. In order to find the optimal performer, four different endonucleases, two of them salt tolerant, were evaluated in downstream processing of recombinant measles virus. Endonuclease treatment was performed under optimal temperature conditions after clarification and before the purification by flow-through chromatography with a core shell chromatography medium: Capto™ Core 700. Virus infectivity was measured by TCID50. DNA and histone presence in process and purified samples was determined using PicoGreen™ assay and Western blot analysis using an anti-histone antibody, respectively. All tested endonucleases allowed the reduction of DNA content improving product purity. The salt-tolerant endonucleases SAN and M-SAN were more efficient in the removal of chromatin compared with the non-salt-tolerant endonucleases Benzonase® and DENARASE®. Removal of chromatin using M-SAN was also possible without the addition of extra salt to the cell culture supernatant. The combination of the endonuclease treatment, using salt-tolerant endonucleases with flow-through chromatography, using core-shell particles, resulted in high purity and purification efficiency. This strategy has all features for a platform downstream process of recombinant measles virus and beyond.