Steric Exclusion Chromatography Research Articles

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis.

By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have beenimproved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 1011 TCID50 /Lbioreactor /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

Process intensification for the production of yellow fever virus-like particles as potential recombinant vaccine antigen.

Yellow fever (YF) is a life-threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000-60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg-derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus-like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence-activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken-tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3-fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one-step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D-structure of the purified VLPs.

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles

BackgroundInfections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.ResultsIn the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E−2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.ConclusionIn summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

Melt strengthening of polylactic acid and its blends: Shear and elongation rheological investigations of the forming process

Polylactic acid (PLA) can be a good alternative to petroleum-based polymers thanks to its organic origin and its biodegradability. This study introduces some promising routes for enhancing the processability of PLA, which presents several challenges due mainly to the poor shear and elongation properties of this biopolymer. To our knowledge, this is the first paper dedicated to an investigation of foaming and/or blown extrusion of PLA that focuses on structural, rheological and thermomechanical properties. Two main routes were selected: (i) the modification of its structural, rheological and thermomechanical properties and (ii) blending the PLA with another ductile, thermoplastic biopolymer such as poly (butylene adipate-co-terephthalate) (PBAT) or polyamide (PA11). Various formulations of PLA with multifunctionalized epoxy, nucleating agents and plasticizer were prepared and characterized on the basis of their linear viscoelasticity and extensional properties. The balance of chain extension and branching was also investigated using solution viscosimetry, steric exclusion chromatography (SEC) and rheology (shear and elongation rheology). On one hand, a batch foaming process assisted by supercritical CO2 was carried out. The influence of the foaming parameters, the extent of chain modification and the contribution of crystallization to cell morphology were all evaluated. Based on these parameters, structures ranging from micro to macro-cellular-cell were obtained. On the other hand, the stability maps of blown extrusion for neat and modified PLA were established at different die temperatures. We succeeded in greatly enhancing the blown extrusion windows of PLA, achieving high blow-up ratio (BUR) and take-up ratio (TUR) values. We were able to demonstrate that faster kinetics of crystallization can also be reached for chain-extended and branched PLA formulated with adequate amounts of nucleating agents and plasticizers. Through this work, blown films with intriguing thermomechanical and mechanical properties were produced using an optimal formulation for PLA.

The Moroccan Pomegranate: An Underrated Source of Tannins Extracts and Natural Antimicrobials from Juice Processing Byproducts

Purpose The pomegranate juice manufacturing industry produces vast quantities of non-edible portions of fruit as a by-product. The rind is a good source of many beneficial functional components, especially polyphenols and in particular tannins. This research was undertaken to expose the presence of active substances, including tannin compounds, in the crude extracts (aqueous (AE) and methanolic (ME)) and their fractions increasing in polarity, from Moroccan pomegranate rind samples. Methods To describe the molecular distribution of tannin content in crude extracts, steric exclusion chromatography (SEC) on Sephadex gel 50 was carried out. In addition, their antimicrobial properties were tested using agar diffusion, dispersion, and microdilution methods against the bacterial strains Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and the yeast strains Saccharomyces cerevisiae and Candida tropicalis. Results For both crude extracts, the SEC showed qualitative and quantitative differences in tannin polymerization. Diameters of inhibition zones (DIZ) obtained against bacterial species ranged from 20.6 to 30.3 mm for ME, from 10.8 to 15.3 mm for AE and from 0 to 16.6 mm for fractions. Among the selected fungal cultures, the highest antifungal activity was reported against S. cerevisiae; with an inhibition rate (I %) ranging up to 99.00% for AE. With I % reaching up to 93.35%, the C. tropicalis strain was more sensitive to ME, although the AE has no inhibitory effect on this yeast. For fractions, the I % ranged from 7.03 to 98.03% where a synergistic antifungal effect was observed between fractions. Conclusion Pomegranate processing by-product is a potential source which could be used as natural preservatives in food industry and non-toxic matrices replacing hazardous materials in surface treatment.

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography.

We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing AAV particles with polyethylene glycol (PEG) and feeding the mixture onto a chromatography filter unit. The purified AAV particles are recovered by flushing the unit with a solution lacking PEG. SXC is an inexpensive single-use method that permits to concentrate, purify, and re-buffer AAV particles with yields >95% and >80% impurity clearance. SXC could theoretically be employed at industrial scales with units of nearly 20 m2.

Characterization and valuable use of Calotropis gigantea seedpods as a biosorbent of methylene blue

In this work, powdered Calotropis gigantea seedpods were characterized and used as biosorbents of methylene blue dye from aqueous solution. FT-IR spectroscopy demonstrated functional groups characteristics of cellulose. Steric exclusion chromatography donated an average molecular weight of 230 kg/mol of the biopolymer. The polymolecularity index value (1.95) proved the good homogeneity of the polysaccharide. Scanning electron microscopy features displayed a homogenous morphology and porous structure. X-ray diffraction patterns showed peaks characteristics of cellulose and non-cellulose compositions. Thermogravimetric analysis/differential thermal analysis displayed exothermal decompositions at 316.9 °C and 456 °C. The maximum biosorption capacity of methylene blue was 88.36 mg/g at pH = 6, time = 60 min, and T = 21 °C. The level was comparable to some other studied agricultural wastes. The adsorption mechanism followed pseudo-second-order and Freundlich models. As it is abundant, available, low-cost, and easily recovered from solution, C. gigantea seedpods could be used as an effective biomaterial for the removal of organic pollutants from contaminated waters. Novelty statement: An abundant, available, and low-cost Calotropis gigantea seedpod was used, for the first time, as an effective biomaterial for the biosorption of organic pollutants. The biosorption level was found to be comparable to some other agricultural wastes studied previously in the literature.

Microgel structure-driven linear and non-linear mechanical properties of self-assembled microgel films

Soft self-supported cohesive films were formed by self-assembly of pH- and thermo-responsive P(MEO2MA-co-OEGMA-co-MAA) microgels without any preparation and/or post-treatments. Linear and non-linear mechanical properties of those films were characterized respectively by dynamic mechanical measurements at low strain and uniaxial extensional tests at high deformation. A relationship between the microstructure of microgels and the mechanical strength of microgel-based films was successfully established. Compared to microgels crosslinked with Oligo(ethylene glycol) diacrylate (OEGDA), microgels with N,N-methylenebisacrylamide (MBA) demonstrate a less crosslinked structure but especially a looser shell which enables the particles to better interpenetrate between one another and resist to higher deformation. Novel films were also designed by controlled mixing microgels and the synthesis side-product, namely water-soluble polymer, WSP, in a ratio with WSP content till 100 %. Those films demonstrated promising mechanical properties due to the structural characteristics of the later. The later act as a lever to tune the elastic modulus of films without diminishing their resistance at strain thanks to its high molar mass and crosslinked structure revealed by Steric Exclusion Chromatography (SEC) measurements.

OP7, a novel influenza A virus defective interfering particle: production, purification, and animal experiments demonstrating antiviral potential

The novel influenza A virus (IAV) defective interfering particle “OP7” inhibits IAV replication in a co-infection and was previously suggested as a promising antiviral agent. Here, we report a batch-mode cell culture-based production process for OP7. In the present study, a seed virus containing standard virus (STV) and OP7 was used. The yield of OP7 strongly depended on the production multiplicity of infection. To inactivate infectious STV in the OP7 material, which may cause harm in a potential application, UV irradiation was used. The efficacy of OP7 in this material was preserved, as shown by an in vitro interference assay. Next, steric exclusion chromatography was used to purify and to concentrate (~ 13-fold) the UV-treated material. Finally, administration of produced OP7 material in mice did not show any toxic effects. Furthermore, all mice infected with a lethal dose of IAV survived the infection upon OP7 co-treatment. Thus, the feasibility of a production workflow for OP7 and its potential for antiviral treatment was demonstrated.Key points• OP7 efficacy strongly depended on the multiplicity of infection used for production• Purification by steric exclusion chromatography increased OP7 efficacy• OP7-treated mice were protected against a lethal infection with IAV

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine

There is a large unmet need for a prophylactic hepatitis C virus (HCV) vaccine to control the ongoing epidemic with this deadly pathogen. Many antiviral vaccines employ whole viruses as antigens. For HCV, this approach became feasible following the development of infectious cell culture systems for virus production. However, the lack of efficient downstream processes (DSP) for HCV purification poses a roadblock for the development of a whole virus vaccine. Using cell culture-derived genotype 1a HCV we developed a scalable and efficient DSP train, employing commonly used clarification and ultrafiltration techniques, followed by two membrane-based chromatography steps. For virus capture, steric exclusion chromatography using cellulose membranes was established, resulting in a virtually complete virus recovery with > 99% protein and 84% DNA depletion. Virus polishing was achieved by sulphated cellulose membrane adsorbers with ~ 50% virus recovery and > 99% protein and 90% DNA depletion. Additional nuclease digestion resulted in 99% overall DNA depletion with final DNA concentrations of 2 ng/mL. Process results were comparable for cell culture-derived HCV of another major genotype (5a). This study provides proof-of-concept for establishment of an efficient and economically attractive DSP with potential application for production of an inactivated whole virus vaccine against HCV for human use.

Upstream and Downstream Processes for Viral Nanoplexes as Vaccines.

The increasing medical interest in viral nanoplexes, such as viruses or virus-like particles used for vaccines, gene therapy products, or oncolytic agents, raises the need for fast and efficient production processes. In general, these processes comprise upstream and downstream processing. For the upstream process, efficiency is mainly characterized by robustly achieving high titer yields, while reducing process times and costs with regard to the cell culture medium, the host cell selection, and the applied process conditions. The downstream part, on the other hand, should effectively remove process-related contaminants, such as host cells/cell debris as well as host cell DNA and proteins, while maintaining product stability and reducing product losses. This chapter outlines a combination of process steps to successfully produce virus particles in the controlled environment of a stirred tank bioreactor, combined with a platform-based purification approach using filtration-based clarification and steric exclusion chromatography. Additionally, suggestions for off-line analytics in terms of virus characterization and quantification as well as for contaminant estimation are provided.

A scalable downstream process for the purification of the cell culture-derived Orf virus for human or veterinary applications

The large demand for safe and efficient viral vector-based vaccines and gene therapies against both inherited and acquired diseases accelerates the development of viral vectors. One outstanding example, the Orf virus, has a wide range of applications, a superior efficacy and an excellent safety profile combined with a reduced pathogenicity compared to other viral vectors. However, besides these favorable attributes, an efficient and scalable downstream process still needs to be developed. Recently, we screened potential chromatographic stationary phases for Orf virus purification. Based on these previous accomplishments, we developed a complete downstream process for the cell culture-derived Orf virus. The described process comprises a membrane-based clarification step, a nuclease treatment, steric exclusion chromatography, and a secondary chromatographic purification step using Capto® Core 700 resin. The applicability of this process to a variety of diverse Orf virus vectors was shown, testing two different genotypes. These studies render the possibility to apply the developed downstream scheme for both genotypes, and lead to overall virus yields of about 64 %, with step recoveries of >70 % for the clarification, and >90 % for the chromatography train. Protein concentrations of the final product are below the detection limits, and the final DNA concentration of about 1 ng per 1E + 06 infective virus units resembles a total DNA depletion of 96–98 %.

Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector

In recent years, the Orf virus has become a promising tool for protective recombinant vaccines and oncolytic therapy. However, suitable methods for an Orf virus production, including up- and downstream, are very limited. The presented study focuses on downstream processing, describing the evaluation of different chromatographic unit operations. In this context, ion exchange-, pseudo-affinity- and steric exclusion chromatography were employed for the purification of the cell culture-derived Orf virus, aiming at a maximum in virus recovery and contaminant depletion. The most promising chromatographic methods for capturing the virus particles were the steric exclusion- or salt-tolerant anion exchange membrane chromatography, recovering 84 % and 86 % of the infectious virus. Combining the steric exclusion chromatography with a subsequent Capto™ Core 700 resin or hydrophobic interaction membrane chromatography as a secondary chromatographic step, overall virus recoveries of up to 76 % were achieved. Furthermore, a complete cellular protein removal and a host cell DNA depletion of up to 82 % was possible for the steric exclusion membranes and the Capto™ Core 700 combination.The study reveals a range of possible unit operations suited for the chromatographic purification of the cell culture-derived Orf virus, depending on the intended application, i.e. a human or veterinary use, and the required purity.

Membrane-based steric exclusion chromatography for the purification of a recombinant baculovirus and its application for cell therapy

The continuously increasing potential of stem cell treatments for various medical conditions has accelerated the need for fast and efficient purification techniques for individualized cell therapy applications. Genetic stem cell engineering is commonly done with viral vectors like the baculovirus. The baculovirus is a safe and efficient gene transfer tool, that has been used for the expression of recombinant proteins for many years. Its purification has been based mainly on ion exchange matrices. However, these techniques impair process robustness, if different genetically modified virus particles are applied. Here, we evaluated the membrane-based steric exclusion chromatography for the purification of insect cell culture-derived recombinant Autographa californica multicapsid nucleopolehydroviruses for an application in cell therapy. The method has already proven to be a powerful tool for the purification of Influenza A virus particles, using cellulose membranes.Aside from the aforementioned cellulose, we evaluated alternative stationary phases, such as glass fiber and polyamide membranes. The highest dynamic binding capacitiy was determined for cellulose with 5.08E + 07 pfu per cm² membrane. Critical process parameters were optimized, using a design of experiments (DoE) approach. The determined process conditions were verified by different production batches, obtaining a mean virus yield of 91% ± 6.5%. Impurity depletion was >99% and 85% for protein and dsDNA, without nuclease treatment. Due to the method’s specificity, its application to other baculoviruses, with varying surface modifications, is conceivable without major process changes. The physiological buffer conditions enable a gentle handling of the virus particles without decreasing the transduction efficacy. The simple procedure with sufficient impurity removal enables the substitution of time-consuming ultra centrifugation steps and can serve as a first process unit operation to obtain higher purities.

Urinary albumin measurement methods.

The quantification of urine albumin is a common practice in Medical Biology laboratories. It allows the assessment of renal injury in common pathologies and many studies have confirmed its role in the diagnosis and prognosis of these disorders. The physicochemical characteristics of albumin in the urine, very different from those in the blood, do not allow the use of the same standardized assay techniques for the blood albumin determination and make it difficult its quantification. Indeed, because of a physiological fragmentation phenomenon, urinary albumin is present in the urine as various small specific peptides. We will present here the main methods of determination of albumin in the urine, which are immuno-turbidimetric and immuno-nephelometric methods, high performance liquid chromatography with steric exclusion and liquid chromatography coupled with mass spectrometry. Currently, immunoanalysis techniques are the most used and are not standardized; large bias can be found between the different kits. This observation calls for a standardization of its determination in the urine.

Application of steric exclusion chromatography on monoliths for separation and purification of RNA molecules

Steric exclusion chromatography (SXC) is a method for separation of large target solutes based on their association with a hydrophilic stationary phase through mutual steric exclusion of polyethylene glycol (PEG). Selectivity in SXC is determined by the size or shape (or both) of the solutes alongside the size and concentration of PEG molecules. Elution is achieved by decreasing the PEG concentration. In this study, SXC applicability for the separation and purification of single-stranded (ss) and double-stranded (ds) RNA molecules was evaluated for the first time. The retention of ssRNA and dsRNA molecules of different lengths on convective interaction media (CIM) monolithic columns was systematically studied under variable PEG-6000 and NaCl concentrations. We determined that over 90% of long ssRNAs (700–6374 nucleotides) and long dsRNAs (500–6374 base pairs) are retained on the stationary phase in 15% PEG-6000 and ≥0.4 M NaCl. dsDNA and dsRNA molecules of the same length were partially separated by SXC. Separation of RNA molecules below 100 nucleotides from longer RNA species is easily achieved by SXC. Furthermore, SXC has the potential to separate dsRNAs from ssRNAs of the same length. We also demonstrated that SXC is suitable for the enrichment of ssRNA (PRR1 bacteriophage) and dsRNA (Phi6 bacteriophage) viral genomes from contaminating cellular RNA species. In summary, SXC on CIM monolithic columns is an appropriate tool for rapid RNA separation and concentration.

Steric exclusion chromatography for purification of cell culture-derived influenza A virus using regenerated cellulose membranes and polyethylene glycol

Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration. In this study, a 75cm2 cellulose membrane adsorber was used for the purification of a clarified and inactivated influenza A virus broth produced in a 5L bioreactor using suspension Madin Darby canine kidney cells. Product recovery was above 95% based on hemagglutination activity and single radial immunodiffusion assays. Maximum depletion of double stranded host cell DNA and total protein was 99.7% and 92.4%, respectively. Purified virus particles showed no aggregation with a monodisperse peak around 84nm. 250mL of the clarified inactivated virus broth was purified within 40min. The surface area productivity based on the recovery of the viral hemagglutinin antigen was 28–50mgm−2h−1 depending on the feed and loading conditions.

Extraction and characterization of pectic substances in Myrciaria cauliflora (Jaboticaba sabará) fruit

The jaboticaba is a fruit that is native to Brazil, which has antioxidant activity and is known to have levels of bioactive compounds. This fruit is a berry with a lilac-coloured husk and white flesh; sweet and acid, which justifies its high levels of fresh consumption. The aim of this study was to contribute to the study of a new raw material in small agribusinesses. Jaboticaba- Sabara variety- was fractionated into three parts for pectin analysis: the skin, pulp and juice. The pectic substances of the jaboticaba were analysed. The degree of methoxylation was determined by titration and infrared spectroscopy (FTIR). Pectin extracted from the skin had a higher degree of esterification. The macromolecular composition, determined by high pressure steric exclusion chromatography (HPSEC), discriminated the pectins of high molar mass (skin) and low molecular weight (juice and pulp). The pectin from all the fractions presented homogalacturonan segments with a high degree of esterification, and ramnogalacturan side chains composed of galactose and arabinose. The jaboticaba pectins presented a high content of arabinose in the skin, whilst galactose was only found in traces in the juice, showing different proportions of monosaccharides, similar to commercial samples of apple pectin.

Two-step syneretic formation of highly porous morphology during copolymerization of hydroxyethyl methacrylate and ethylene glycol dimethylacrylate

Large pore volume and pore size ranges were achieved in 2-hydroxyethyl methacrylate (HEMA)/ethylene glycol dimethylacrylate (EGDMA) copolymers prepared by microsyneretic phase separation in free-radical copolymerization of comonomers in the presence of a tenfold volume of porogenic solvents. A mixture of methanol, i.e. good swelling solvent for poly(HEMA), and tetrahydrofuran, i.e. good swelling solvent for poly(EGDMA), was used as porogens. The morphology of the as-prepared copolymers was assessed in their native, expanded state using inverse steric exclusion chromatography (ISEC), which enabled to get a much better insight into the mechanism of pore formation than a conventional approach based on the evaluation of the morphology of dried materials. Two porosity levels with distinct pore volumes and sizes could be distinguished depending on the crosslinking degree and the MeOH/THF ratio. Their formation could be explained by a two-step syneretic phase separation during the free-radical copolymerization, namely χ-induced syneresis for the larger pore sizes and ν-induced syneresis for the smaller pore sizes Due to their high crosslink density, the porosity of the resulting polymers do not rely on swelling, and it was virtually independent on the nature of surrounding solvent.