Chromatographic Media Research Articles

The checkerboard model for the Eddy-dispersion in Laminar flows through porous media. Part II: Application to ordered and disordered 2-D flow systems

We report on a series of high-accuracy computational fluid dynamics band broadening simulations in three different 2-D flow systems: a 2-D pillar array and 2-D lumped packed bed geometries with different checkerboard velocity bias patterns. These media display a local maximum in the relationship between the eddy-dispersion plate height and the mobile phase velocity. The occurrence of such a dispersion maximum has not been reported before but appears to be a characteristic of regular chromatographic media with alternating velocity bias, at least in 2-D geometries. This newly observed behavior can be fully understood and modelled using the checkerboard model established in part I of the present study.

Mechanism of bio-macromolecule denaturation on solid-liquid surface of ion-exchange chromatographic media – A case study for inactivated foot-and-mouth disease virus

Destruction of assembly structures has been identified as a major cause for activity loss of virus and virus-like particles during their chromatographic process. A deep insight into the denaturation process at the solid-liquid interfaces is important for rational design of purification. In this study, in-situ differential scanning calorimetry (DSC) was employed to study the dissociation process of inactivated foot-and-mouth disease virus (FMDV) during ion exchange chromatography (IEC) at different levels of pH. The intact FMDV known as 146S and the dissociation products were quantified by high performance size exclusion chromatography (HPSEC) and the thermo-stability of 146S on-column was monitored in-situ by DSC. Serious dissociation was found at pH 7.0 and pH 8.0, leading to low 146S recoveries of 12.3% and 43.7%, respectively. The elution profiles from IEC and HPSEC combined with the thermal transition temperatures of 146S dissociation (Tm1) from DSC suggested two denaturation mechanisms that the 146S dissociation occurred on-column after adsorption at pH 7.0 and during elution step at pH 8.0. By appending different excipients including sucrose, the improvement of 146S recovery and reduced dissociation was found highly correlated to increment of 146S stability on-column detected by DSC. The highest recovery of 99.9% and the highest Tm1 of 54.49 °C were obtained at pH 9.0 with 20% (w/v) sucrose. According to chromatographic behaviors and Tm1, three different dissociation processes in IEC were discussed. The study provides a perspective to understand the denaturation process of assemblies during chromatography, and also supplies a strategy to improve assembly recovery.

A Flow-Through Chromatographic Strategy for Hepatitis C Virus-Like Particles Purification

Biopharmaceuticals are currently becoming one of the fastest growing segments of the global pharmaceutical industry, being used in practically all branches of medicine from disease treatment to prevention. Virus-like particles (VLP) hold tremendous potential as a vaccine candidate due to their anticipated immunogenicity and safety profile when compared to inactivated or live attenuated viral vaccines. Nevertheless, there are several challenges yet to be solved in the development and manufacturing of these products, which ultimately can increase time to market. Suchlike virus-based products, the development of a platform approach is often hindered due to diversity and inherent variability of physicochemical properties of the product. In the present work, a flow-through chromatographic purification strategy for hepatitis C VLP expressed using the baculovirus-insect cell expression system was developed. The impact of operational parameters, such as residence time and ionic strength were studied using scaled-down models and their influence on the purification performance was described. The flow-through strategy herein reported made use of radial-flow chromatography columns packed with an anion exchanger and was compared with a bind and elute approach using the same chromatography media. Overall, by selecting the optimal operational setpoints, we were able to achieve higher VLP recoveries in the flow-through process (66% versus 37%) with higher removal of DNA, baculovirus and host-cell protein (92%, 99% and 50% respectively).

A Review on Comparative study of HPLC and UPLC

Recent advancements in pharmaceutical analysis have made available chromatographic media with a 1.7 μm particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed Ultra performance liquid chromatography (UPLC) using sub 2 micron particles and very high pressure (up to 100 MPa is possible in UPLC system) has demonstrated improvement in method sensitivity, resolution and speed compared with conventional HPLC. The UPLC system allows decreasing analysis time up to nine times compared to chromatographic system using 5 μm size particle packed analytical columns. In comparison with 3μm size particle packed analytical columns analysis time shortened about three times. The present review paper differentiates HPLC and UPLC, analytical method validation, applications, advantages and disadvantages of HPLC and UPLC.

Affinity Membranes and Monoliths for Protein Purification.

Affinity capture represents an important step in downstream processing of proteins and it is conventionally performed through a chromatographic process. The performance of this step highly depends on the type of matrix employed. In particular, resin beads and convective materials, such as membranes and monoliths, are the commonly available supports. The present work deals with non-competitive binding of bovine serum albumin (BSA) on different chromatographic media functionalized with Cibacron Blue F3GA (CB). The aim is to set up the development of the purification process starting from the lab-scale characterization of a commercially available CB resin, regenerated cellulose membranes and polymeric monoliths, functionalized with CB to identify the best option. The performance of the three different chromatographic media is evaluated in terms of BSA binding capacity and productivity. The experimental investigation shows promising results for regenerated cellulose membranes and monoliths, whose performance are comparable with those of the packed column tested. It was demonstrated that the capacity of convective stationary phases does not depend on flow rate, in the range investigated, and that the productivity that can be achieved with membranes is 10 to 20 times higher depending on the initial BSA concentration value, and with monoliths it is approximately twice that of beads, at the same superficial velocity.

Attenuated total reflection fourier transform infrared spectroscopy based methods for identification of chromatography media formulations used in downstream processes

Chromatographic media play a crucial role in the downstream processing of biotechnology products. The physical and chemical properties of these processing aids are mostly monitored by expensive and time‐consuming preparative tests, but spectroscopic techniques may also be used to measure chromatographic media samples.In this study, chromatographic media formulations used in downstream processes were investigated using attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. Samples were measured both in original suspension form and after drying to examine the possibilities of a potential spectroscopic method without sample preparation. Principal component analysis (PCA) was employed to identify the spectral differences among the formulations with distinct support matrices and functional groups and soft independent modeling of class analogy (SIMCA) was performed to creating classification models for identification of chromatography media. To increase the number of samples in the SIMCA, simulated spectra were generated based on the experimental spectra.PCA models indicated that spectra of samples in original suspension form and after drying contained similar information about the chemical properties of chromatographic media samples. Moreover, during the classification of spectra based on SIMCA, both measurement techniques gave high sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) results.These results show that ATR FT-IR could be applied as a simple alternative method for monitoring the chromatography media samples. This technique is also feasible without sample preparation. Thereby the multi-hours drying steps may be omitted, the measurements can be performed in a few minutes, and the potential effects of sample preparations can be eliminated.

Evaluating the ion exchange chromatography for matrix-assisted PEGylation and purification of consensus interferon.

Scientists have implemented protein-PEGylation technology for boosting-up the pharmacokinetics and stability of recombinant therapeutic proteins. In the present study, (a) matrix-assisted PEGylation was compared with solution-phase PEGylation and (b) matrix-assisted PEGylation was performed with different ion exchange resins for impact of chromatography medium on yield and purity of PEGylated product. DEAE Sepharose CL 6B, DEAE Fracto gel, and Macro cap Q ion exchange chromatography medium were compared for on column PEGylation and purification of cIFN. A MSC-PEG of 12.0 KDa was selected. cIFN was bound to ion exchange medium, and PEG solution was passed through resin for 180 Min, and protein was eluted by sodium chloride linear gradient. Yield and purity for mono-PEGylated cIFN with Macro cap Q matrix was 75% and 99%, respectively, whereas for DEAE Sepharose was 45% and 60%. DEAE Fracto gelTM purity was 85% with 50% yield of mono-PEGylated cIFN. Further investigation of in vitro biological activities demonstrated that about 30% antiviral activity was reduced as compared to unmodified cIFN. However, thermal stability was significantly improved. The present study proved that matrix-assisted PEGylation can improve the yield and purity of mono-PEGylated product, and Macro Cap resin provided the highest yield of a homogeneous product. In present study, (a) matrix-assisted PEGylation was compared with solution-phase PEGylation and (b) matrix-assisted PEGylation was performed with different ion exchange resins for impact of chromatography medium on yield and purity of PEGylated product. Matrix-assisted PEGylation increases the yield of mono-PEGylated product and further Macro CapTM produced highest yield and purity of PEGylated cIFN.

Computational fluid dynamic (CFD) simulation of a cuboid packed-bed chromatography device

Flow non-uniformity is a major problem with process chromatography columns which typically have small bed height to diameter ratios. In recent publications, we have proposed box-shaped or cuboid packed-bed devices as efficient alternative to process columns for carrying out high-resolution chromatographic separations. Cuboid packed-bed devices show superior performance in terms of number of theoretical plates, peak width, and peak resolution in multicomponent separations. This paper attempts to explain this based on computational fluid dynamic (CFD) simulations of a cuboid packed-bed device and its equivalent column, i.e. having the same bed height and area of cross-section, and packed with the same chromatographic media. The radial velocity in the column headers decreased very rapidly from the axis to the periphery resulting in significant variation in average velocity along the different flow paths within the column. By contrast, the velocity decreased linearly in the top channel and increased linearly in the bottom channel of a cuboid packed-bed device, resulting in significantly lower variation in the average velocity. Simulated flow-through peaks obtained with the cuboid packed-bed device using dextran as a macromolecular and sodium chloride as a low molecular weight tracer were significantly sharper and more symmetric. Experimental results were in good agreement with those obtained by simulation. Overall, the superior performance of the cuboid packed-bed device could be primarily attributed to the narrower solute residence time distribution resulting from greater flow uniformity.

A frontal analysis combined with a simultaneous chromatographic analysis of macromolecules using a single chromatographic system

A chromatographic system was adapted to allow monitoring of eluent of preparative column via absorbance and with the chromatographic analysis of the target macromolecule on the same chromatographic system. The proposed approach was tested on important macromolecules, such as monoclonal antibodies, monoclonal antibody aggregates and plasmid DNA (pDNA). A frontal analysis was made on the preparative column, while a chromatographic on-line analysis was performed by sequentially injecting the preparative column outlet on a convection-based analytical column, operating on the same chromatographic system. Cation and/or anion exchangers were used as the chromatographic media (along with a protein A), depending on the sample to be purified. The method was found to be robust and reproducible. To adjust the limit of detection, an algorithm varying the number of injections was used, enabling accurate monitoring of an early breakthrough for concentrations below 1% of the feed concentration. The accuracy varies according to the applied flow rate, but it is typically in the range of few percent, or even below. Due to its simplicity and flexibility, the proposed method can be easily adapted to a pharmaceutical environment.

A new reliable test for crossmatching: microplate hydrogel immunoassay technology

To provide a novel assay to detect incomplete antibodies in the crossmatching test. There is a requirement in China that both major and minor crossmatch tests are required. Among all methods of crossmatching, the tube anti-human globulin test requires tedious washing steps and is time-consuming, whereas the microcolumn gel immunoassay anti-human globulin test is susceptible to sample quality. The process of the microplate hydrogel immunoassay anti-human globulin test involves the use of our patented hydrogel chromatography medium and U-bottom microplates pre-coated with goat anti-human globulin (AHG). A mixture of red blood cells (RBCs) and serum is centrifuged through the hydrogel under precise conditions. In incompatible reactions, the sensitised RBCs are captured by the pre-coated AHG and form a layer over the bottom of the well, whereas in compatible reactions, the unbound RBCs form a button at the bottom of the well. The sensitivity of this new approach and the performance when testing old specimens were evaluated. This approach was more convenient and slightly more sensitive than the tube anti-human globulin test and was superior to the microcolumn gel immunoassay when testing old specimens. In general, this assay is suitable for the routine clinical use of crossmatching to detect incomplete antibodies.

Nanoparticle-doped polystyrene/polyacrylonitrile nanofiber membrane with hierarchical structure as promising protein hydrophobic interaction chromatography media

Abstract Hydrophobic interaction chromatography (HIC) as an indispensable method for protein purification has attracted considerable attentions of researchers as well as biopharmaceutical industries. However, the operation pressure-drop and flow rate of the typical HIC porous resin beads are less than satisfactory. To address these issues, composite nanofibrous membranes (CNFMs) with controllable wettability have been developed by multi-component electrospinning of polystyrene/polyacrylonitrile (PS/PAN), and further by regulating the solution properties and introducing SiO 2 nanoparticles in, CNFMs with proper wettability and large surface area of 23.6 m−2 g−1 were fabricated. The obtained CNFMs showed a high static adsorption capacity of 78 mg g−1 and fast adsorption process within 30 min. Additionally, a high saturation dynamic binding capacity of 39.6 mg ml−1 was obtained under a low pressure-drop of 200 Pa, which surpassed the commercial HIC membrane media. Moreover, these membranes have a selectivity toward the protein with higher hydrophobicity , and proteins adsorbed on the membranes can be eluted with 10 wt% ethanol aqueous solution. Therefore, such a successful fabrication of nanofibrous membranes with outstanding hydrophobic protein adsorption performance could provide a promising platform to prepare economical and inspiring HIC media in the future.

Elaborate design of ethylene vinyl alcohol (EVAL) nanofiber-based chromatographic media for highly efficient adsorption and extraction of proteins

The development of chromatographic media with superb adsorption capacity and large processing throughput is of great importance for highly efficient protein adsorption and separation, yet still faces a huge challenge. Herein, a new kind of butane tetracarboxylic acid (BTCA) functionalized ethylene vinyl alcohol (EVAL) nanofibrous membranes (BTCA@EVAL NFM)-based chromatographic media is fabricated, for the first time, by combining blend electrospinning technique with in-situ modification technology. The resulting BTCA@EVAL NFM possesses an enhanced equilibrium protein adsorption capability (716 mg g−1), a high saturated dynamic protein binding capacity (490 mg g−1), and a distinctive selectivity towards positively charged proteins, which are attributed to the synergistic effects of the hydrophilic EVAL nanofibrous matrix and the plentiful carboxyl groups introduced by BTCA. Besides, benefiting from its stable physical and chemical structures, the membrane also presents excellent acid and alkaline resistance as well as good reusability. Significantly, the BTCA@EVAL NFM can directly extract lysozyme from egg white with a relatively large capture capability of 353 mg g−1, highlighting its superb potential practicability. We sincerely hope that this new design concept and the highly effective nanofiber-based chromatographic media can provide some guidance for the further development of bio-separation and purification.

An annular-flow, hollow-fiber membrane chromatography device for fast, high-resolution protein separation at low pressure

High-resolution protein separation is currently carried out using columns packed with ultrafine particulate chromatographic media which can only be operated at very high pressures. This restricts the flow rate and thereby the speed of separation. A membrane with ultrafine pores shares the same mass transport advantages as a bed of ultrafine particles. However, application of membrane chromatography has largely been restricted to low-resolution separation applications, primarily due to limitations associated with poorly designed devices. In this paper, we describe a novel annular-flow hollow-fiber membrane chromatography (AHMC) device, suitable for fast, high-resolution protein separation. The AHMC device was designed primarily from a fluidics perspective to ensure a narrow solute residence time distribution, a low dead-volume, and minimal fluid back-mixing. Using this device, it was possible to separate a ternary protein mixture in less than a minute, at less than 0.3 MPa backpressure. The high-resolution separation capability of AHMC was further demonstrated through its use for the separation of bovine serum albumin (BSA) dimer from the monomeric form of the protein. This was achieved in less than 3 min which was considerably faster than other reported techniques for protein aggregates separation.

Fabrication of rigid and macroporous agarose microspheres by pre-cross-linking and surfactant micelles swelling method

A novel combined method of pre-cross-linking and surfactant micelles swelling was proposed in this study to fabricate highly cross-linked and macroporous agarose (HMA) microspheres. Agarose was chemically modified by allylglycidyl ether (AGE) as heterobifunctional cross-linker via its active glycidyl moieties before gel formation and pre-cross-linking was achieved. By this means, the effective concentration of cross-linker presented in agarose gel increased significantly, and thus cross-linking with a high-efficiency was achieved. Further to enhance the intraparticle mass transfer of agarose microspheres, the surfactant micelles swelling method was utilized to create interconnected macropores. Under the optimal condition, HMA microspheres with homogeneous reticular structure and pore size of hundreds nanometers were successfully prepared. They exhibited a low backpressure with a flow velocity as high as 1987 cm/h, which was much higher than that of commercial Sepharose 4 F F. HMA microspheres were then derivatized with carboxymethyl (CM) groups and applied in ion-exchange chromatography. As expected, CM-HMA column separated model proteins effectively even at a flow velocity three times higher than that of commercial CM-4 F F. Visualization of dynamic protein adsorption by confocal laser scanning microscope (CLSM) revealed that the intraparticle mass transfer of CM-HMA microspheres was intensified due to its macroporous structure. All of the results indicated the newly developed agarose microspheres were a promising medium for high-speed chromatography.

Polymer-grafted chromatography media for the purification of enveloped virus-like particles, exemplified with HIV-1 gag VLP

Polymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification. We used HIV-1 gag VLPs with a diameter of 100–200 nm as a model to demonstrate that polymer-grafted anion exchangers are suitable for the purification of bionanoparticles. The equilibrium binding capacity was 1 × 1013 part/mL resin. Moderate salt concentration up to 100 mM NaCl did not affect binding, allowing direct loading of cell culture supernatant onto the column for purification. Dynamic binding capacity at 10% breakthrough, when loading cell culture supernatant, was approximately 6 × 1011 part/mL column; only 1-log lower than for monoliths. Endonuclease treatment of the cell culture supernatant did not increase the dynamic binding capacity, suggesting that dsDNA does not compete for the binding sites of VLPs. Nevertheless, due to simultaneous elution of particles and dsDNA, endonuclease treatment is required to reduce dsDNA contamination in the product. Proteomic analysis revealed that HIV-1 gag VLPs contain different host cell proteins in their cargo. This cargo is composed of conserved proteins and other proteins that vary from one particle population to another, as well as from batch to batch. This process allowed the separation of different particle populations. HIV-1 gag VLPs were directly captured and purified from cell culture supernatant with a total particle recovery in the elution of about 35%. Columns packed with beads can be scaled to practically any dimension and therefore a tailored design of the process is possible.

Preparation and properties of a new alkali-resistant rProtein A chromatographic medium

Preparation and properties of a new alkali-resistant rProtein A chromatographic medium

Identification and characterization of co-purifying CHO host cell proteins in monoclonal antibody purification process

Host cell proteins (HCPs) are process-related impurities derived from the host organism such as Chinese hamster ovary (CHO) cells used for the production of therapeutic mAbs in biopharmaceuticals and potentially pose a risk to patient safety and product efficacy. A number of HCPs have been reported as exceptionally difficult to remove and persist across downstream purification operations into final drug product because they exhibit association with mAbs. Therefore, understanding of HCP impurities and the mAb itself will provide insights into the rational design of efficient downstream process. The aim of this work is to understand mAb interaction with HCPs and identify co-purified HCP subpopulations using two different approaches: (1) Incubation of purified mAb with harvest cell culture fluid (HCCF) from mock-transfected CHO cells (null HCCF) or (2) Immobilization of mAb onto chromatography media followed by incubation with null HCCF. CHO HCP ELISA was used to semi-quantitatively measure the levels of total HCPs. Orthogonal techniques including 2-DE and LC-MS/MS were applied to detect variations in CHO HCP profiles and species. The HCP contents in protein A product pools were significantly higher compared to that in control sample without mAb spiked in and variable HCP levels shown in three different protein A product pools. The majority of HCPs identified by LC-MS/MS in the three protein A product pool showed overlap with the HCP identified in eluate pools from the column immobilized with three different mAbs. The interacting HCPs associated with mAbs were largely involved in catalytic activity. Both approaches demonstrated mAbs bind a common set of HCPs as well as HCPs unique to the mAb.

Rapid Purification of Endotoxin-Free RTX Toxins.

Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).

Removal of Copper in Microdroplets by Ovomucoid Hydrolysates Bound to Reverse-Phase Chromatography Media Within Pipette Tips.

Ovomucoid (OVM) is a protein found in chicken egg white. When it is hydrolyzed by a protease, subtilisin A from Bacillus licheniformis, it possesses Cu2+-chelating activity. In the present work, we demonstrate that the resulting OVM hydrolysates bind to reverse-phase chromatography media in pipette tips and can be applied to remove Cu2+ within microdroplets. 1.4nmol of purified OVM was digested in the presence of 17pmol of subtilisin A at 55°C for 3h. The OVM hydrolysates efficiently removed 2.1 and 2.4nmol of Cu2+ in the droplets by binding to the C4 and C18 chromatography media, respectively. Conversely, 0.6 and 1.0nmol of Cu2+ were removed by the non-digested OVM bound to the C4 and C18 media, respectively. The removal ratio of Cu2+ increased as more OVM was digested by subtilisin A. The digested OVM polypeptides were stained with Cu2+ after they were separated by non-denaturing electrophoresis. These results indicate that OVM hydrolysates bound to chromatography media in a pipette tip can be applied to remove Cu2+ within microdroplets of biological samples.

Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised\xa0on affinity resins

Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics.