Comparative study of thiophilic functionalised matrices for polyclonal F(ab')2 purification

Thiophilic adsorbents have been developed using divinyl sulfone or epoxy activated Streamline quartz base matrix. Their capacity and selectivity for binding polyclonal F(ab')2 fragments generated by whole serum proteolysis was tested. Except for epoxy activated guanidine, all the adsorbents displayed high selectivity for F(ab')2 with dynamic binding capacities ranging from 3 to 10 mg/ml of adsorbent. Thiol immobilised ligands adsorbed more F(ab')2 and the recovery was equal to or more than that from amino immobilised ligands. All adsorbents showed good selectivity for IgG and the dynamic binding capacities were better than for F(ab')2.     

Pressure-Swing Adsorption Using Layered Adsorbent Beds with Different Adsorption Properties: II—Experimental Investigation

An experimental study was conducted on a layered-bed pressure-vacuum-swing adsorption, PVSA, process with adsorbents that differ in their adsorption properties. An oxygen, O₂, PVSA process was employed as an example for investigating how the process performance is affected by bed-layering configuration under different operating conditions for specific purge, product purity, and cycle feature. For two adsorbents with similar nitrogen-to-oxygen, N₂/O₂, selectivity but different N₂ and O₂ capacities, placing the high-capacity adsorbent at the product end and the low-capacity adsorbent at the feed end of the adsorption bed results in a better performance than in the case of reversing the layer positions of those adsorbents. The benefit of placing the adsorbent with higher capacity at the product end becomes more significant at high O₂ product-purity levels. The experimental data obtained in this investigation agree well with simulation results reported earlier.     

Ketoimine modified silica as an adsorbent for gas chromatographic analysis of olefins

New adsorbents for capillary complexation GC have been proposed. The adsorbents contain free ketoimine groups which enable binding copper(II) and chromium(III) chlorides. The adsorbents obtained have been characterised by elemental analysis, differential scanning calorimetry and electron spin resonance. The specific interactions between the adsorbent and adsorbate molecules have been characterised by retention parameters (retention index - I, specific retention volume - Vg, molecular retention index - deltaMe). Attempts were also made at separating mixtures of olefins.     

Adsorptive separation in the enhancement of butene dehydrogenation

Fixed-bed columns containing solid catalysts and adsorbents were employed for simultaneous reaction and separation. The models developed for butene dehydrogenation reaction were validated with experimental data. The model was then employed for variable bed configurations with and without the effect of pressure and vacuum swing reaction (PSR and VSR). The models for the mass and momentum transfer in the catalyst bed and adsorber were solved using orthogonal collocation within the method of lines. The reactor/separator performances were tested for beds with varying numbers of layers of catalysts and adsorbents arranged sequentially. The reaction columns behaved as reactor/separators in series. As the number of layers increased, a homogeneous distribution of the catalyst and adsorbent was approached in the limit. These configurations with variable catalyst/adsorbent distributions were investigated in terms of product purity, selectivity, conversion, recovery and yield. Improved reactor performance was observed with pressure and vacuum swing separation systems and in particular with close to well-mixed reactor/separator configurations.     

Influence of the ionic form of a cation-exchange adsorbent on chromatographic separation of galactooligosaccharides

Separation performance of sodium, lithium, potassium and hydrogen ionic forms of a commercial cation exchanger, Diaion UBK 530, based on a styrene-divinylbenzene copolymer resin support was studied. The adsorbents were applied to the separation of galactooligosaccharides from their mixture with lactose, glucose and galactose. Separation performance of the hydrogen ionic form of the adsorbent was superior to that of other adsorbent forms. A much higher yield of galactooligosaccharides of the requested purity was achieved for this adsorbent form owing to its better kinetic properties. Adsorption properties of individual adsorbents were discussed in relation to possible adsorption mechanisms.     

Mass transfer characteristics of plasmids in monoliths

The hydrodynamic properties and pore-structure of monoliths based on functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) were characterised by pulse response experiments using different probes representing a wide range of molecular mass. On a small scale, band spreading was found to be caused to the extent of more than 90% by extra-column effects. These monoliths have large channel diameters, providing a suitable chromatography adsorbent for processing of large molecules. Dynamic and static binding capacity for plasmid DNA was investigated. For our model plasmid, consisting of 4.9 kbp, a capacity of 7 mg/mL was observed in comparison to 0.3 mg/mL for a conventional medium designed for protein separation. When plasmids were loaded on the monolith a gradual increase in pressure drop was observed. The channels filled up and the cross-sectional area available for liquid flow decreased. Therefore, a higher pressure drop was observed during elution. This is caused by (i) shrinking of the channels as effect of the high salt concentration, (ii) high viscosity of the mobile phase due to high concentration of plasmids, and (iii) an increase of the hydrodynamic radius of the plasmid with salt concentration from 45 nm at 150 mM to 70 nm at 2 M NaCl, as measured by dynamic light scattering. These types of monoliths are considered to be the preferred adsorbents for plasmid separation.     

Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents

A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g^?1), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC‐MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 μg mL^?1. Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step‐purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high‐purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process.     

Effect of phenyl sepharose ligand density on protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

In the large-scale manufacturing and purification of protein therapeutics, multiple chromatography adsorbent lots are often required due to limited absorbent batch sizes or during replacement at the end of the useful column lifetime. Variability in the adsorbent performance from lot to lot should be minimal in order to ensure that consistent product purity and product quality attributes are achieved when a different lot or lot mixture is implemented in the process. Vendors of chromatographic adsorbents will often provide release specifications, which may possess a narrow range of acceptable values. Despite relatively narrow release specifications, the performance of the adsorbent in a given purification process could still vary from lot to lot. In this case, an alternative use test (one that properly captures the lot to lot variability) may be required to determine an acceptable range of variability for a specific process. In this work, we describe the separation of therapeutic protein monomer and aggregate species using hydrophobic interaction chromatography, which is potentially sensitive to adsorbent lot variability. An alternative use test is formulated, which can be used to rapidly screen different adsorbent lots prior to implementation in a large-scale manufacturing process. In addition, the underlying mechanism responsible for the adsorbent lot variability, which was based upon differences in protein adsorption characteristics, was also investigated using both experimental and modeling approaches.     

Pressure-Swing Adsorption Using Layered Adsorbent Beds with Different Adsorption Properties: I—Results of Process Simulation

A simulation study was conducted on layered-bed pressure-swing adsorption, PSA, processes with adsorbents that differ in their adsorption properties. As an example, an oxygen, O₂, vacuum-swing adsorption, VSA, process was analyzed to investigate relationships between process performance and adsorption properties of the adsorbents used. For two adsorbents with identical nitrogen-to-oxygen, N₂/O₂, selectivity but different N₂ and O₂ capacities, placing the high-capacity adsorbent at the product end and the low-capacity adsorbent at the feed end of the adsorption bed gives a better performance than the case of reversing layering of these adsorbents. However, for two adsorbents with different values of N₂/O₂ selectivity but identical N₂ capacity, changing the bed-layer configuration does not show a significant difference in O₂-VSA performance. The advantages of layering a high-capacity adsorbent on product end of the bed are demonstrated by an examination of the N₂-loading difference in a VSA cycle. The modeling study also reveals an effect of cycle features (e.g., equalization step) on the effectiveness of using layered-bed configurations in VSA/PSA processes. It suggests that layering appropriately two adsorbents with different adsorption properties could result in better VSA/PSA-process performance than using a single-layer bed with either of the two adsorbents.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

Improved purification of immunoglobulin G from plasma by mixed‐mode chromatography

Efficient loading of immunoglobulin G in mixed‐mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed‐mode ligand, 4‐(1H‐imidazol‐1‐yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15–64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification.     

High selective purification of flavonoids from natural plants based on polymeric adsorbent with hydrogen-bonding interaction

The efficient purification method of high purity flavonoids from natural plants was reported. A series of polymeric adsorbents with novel structure were synthesized based on the copolymerization of methyl acrylate (MA) and ethylene glycol dimethacrylate (EDGMA). Functional groups, such as ester, amino or amide group, were introduced into the adsorbent matrix, respectively, to produce the hydrogen-bonding interaction and enhance the adsorption selectivity towards flavone compounds. The influences of matrix structure and functional groups of synthesized adsorbents on the adsorption selectivity were investigated. The resins were applied to purify flavonoids in natural plants. It was illuminated that the adsorbent No. 3B with 15% EGDMA content and amide groups performed optimal selectivity to flavone compounds in Scutellaria barbata D.Don, from which the purity of flavonoids in extracts was obtained more than 50%, obviously higher than that from commercial adsorbents. The result of adsorption thermodynamics experiment showed that the isosteric adsorption enthalpy of No. 3B was in the range of 25–30 kJ/mol, which testified that the adsorption mechanism was related to hydrogen-bonding interaction. The method showed its universality via good effects on the purification of total flavonoids from Ginkgo biloba L., Radix puerariae and Hypericum perforatum L.     

Determination of Arsenic by Electrothermal Atomic Absorption Spectrometry Using Arsine Preconcentration on Palladium-Containing Adsorbents

A procedure was developed for the preconcentration of arsine on palladium-containing adsorbents followed by the determination of arsenic by electrothermal atomic absorption spectrometry. Aqueous suspensions of the adsorbent were placed in a graphite furnace at the determination step. The selection of the adsorbent was substantiated; adsorption properties of palladium-containing adsorbents were studied to validate their modifying properties. The absolute and concentration limits of detection for arsenic were 28 pg and 12 ng/L, respectively (sample volume of 100 mL).     

K2CO3活化煤矸石制备活性炭吸附剂

用Ｋ２ＣＯ３化学活化煤矸石制备适用于废水处理的活性炭吸附剂．考察了活化条件对产物的比表面、孔体积及灰分的影响．增加了前处理和后处理步骤以改善产物的性能．最佳条件下获得的活性炭吸附剂ＢＥＴ比表面达１２３６ｍ２／ｇ，孔体积０．６７９ｃｍ３／ｇ．所制得的吸附剂表面是疏水性的，对水溶液中的酚类污染物有良好的吸附性能．     

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni(2+) adsorbent had the greatest capture capacity (6.7 x 10(8) IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.     

高比表面季铵基树脂的结构调控及在三七叶总皂甙纯化中的应用研究

利用Davankov后交联反应,合成了一类兼具高比表面积和高功能基含量的季铵基(—N+(CH)3)吸附树脂,考察了树脂比表面积和功能基含量的调控规律,并将其用于三七叶总皂甙的进一步纯化.结果表明,当树脂比表面积为692m2/g,交换量为2.1mmol/g时,树脂具有最佳的纯化效果,只通过吸附—解吸一步工艺,产品纯度即可从32.0%提高到90%以上,皂甙的回收率高于95%.最后,初步探讨了树脂对皂甙和色素的吸附机理,认为树脂对皂甙的吸附是单纯的疏水性作用力,而对色素的吸附应为疏水-离子交换双重作用的协同效应.     

离子印迹法制备对铜离子(Ⅱ)选择性吸附的碳基材料

通过钨离子印记方法,合成了对铜离子有高选择性吸附性能的碳基材料。 在四乙烯五胺(TEPA)、钨酸铵和葡萄糖摩尔比为1:1:5条件下,经水热法合成的碳基吸附材料能有效地吸附铜离子,对铜离子吸附的最佳pH=5。 在Cd²⁺、Co²⁺、Cu²⁺、Ni²⁺和Zn²⁺摩尔浓度相近体系中,离子印记碳基吸附材料对铜离子的吸附率高达97.2%,而对Cd²⁺、Co²⁺、Ni²⁺、Zn²⁺吸附率分别为20.1%、9.63%、20.3%、13.3%,表明这种离子印记碳基吸附材料对Cu²⁺具有非常优异的选择吸附能力。     

Preparation and evaluation of polymer-coated adsorbents for the expanded bed recovery of protein products from particulate feedstocks

A novel prototype polymer-coated adsorbent (PCA) has been developed for the effective expanded bed recovery of protein products from particulate feedstocks. The adsorbents were manufactured using the three-phase emulsification process by which the selected core phases (anion- and cation-exchangers and a custom-assembled pseudo-affinity adsorbent) were coated by an agarose gel. This new non-stick exterior coating acts as a sieve reducing the non-specific binding of cell and cell debris without diminution of selective capture of target protein from complex feedstocks such as whole microbial broths and cell disruptates. The new coated adsorbents were subjected to physical and hydrodynamical comparison with the performance of their uncoated adsorbents. Hydrodynamic characteristics (e.g. axial dispersion coefficient (D(axl)) and Bodenstein number (B(o))) of PCA demonstrated a marked robustness in the face of biomass loading disrupted yeast cells. In addition, each adsorbent was compared with its uncoated native form during the expanded bed adsorption of one of two intracellular proteins (i.e. glyceraldehyde 3-phosphate dehydrogenase and cytochrome c) from a 20% (ww/v) yeast disruptate. The performance parameters of efficiency of washing, purification factor, turbidity of the eluted product and protein recovery in all analysed cases were favourable to the coated materials. In particular, exploiting PCA reduced significantly undesirable adsorption of cells without significant loss of binding capacity for the target product. The generic application of such adsorbents and their potential for the recovery of target products from complex feedstock is discussed, whilst other application such as the subtractive purification of nanoparticles were detailed in our previous publication.     

A fluorescent high-throughput assay for double-stranded DNA: the RediPlate PicoGreen assay

The fluorescent PicoGreen reagent for detection and quantitation of double-stranded DNA has been adapted for high-throughput screening: the RediPlate PicoGreen double-stranded DNA assay format. In the RediPlate PicoGreen assay format, the PicoGreen reagent is predistributed and co-dried into either 96- or 384-well microplates with the excipient trehalose. The user resuspends the dried reagents upon adding DNA, and measures the resulting fluorescence after a five minute incubation. Replicate fluorescence measurements on nominally identical wells have less than a 5% coefficient of variation. The assay is linear from 5 to 500 ng/ml DNA in a 200 micro l volume. The RediPlate PicoGreen assay format retains the advantages of the original PicoGreen reagent - sensitivity, speed, and specificity - but in a high-throughput format.     

Adsorption Strategy of Plasmid DNA Nanoparticulate: Preparative Purification by a Simple Custom Expanded Bed Column

This paper summarizes the critical examination of the hydrodynamic performance of the NBG expanded bed contactor operated with streamline-DEAE adsorbent under various operating conditions for expanded bed adsorption of plasmid DNA nanoparticles from alkaline lysate. The purification process is not RNase-free. In this study, a rapid and efficient scaleable purification protocol obtaining, plasmid DNA nanoparticles (average size of 40 nm) with a high purity level for use as therapeutic agent in customized NBG expanded bed columns was developed. This technique allows efficient levels of binding to the column media and vector purification without centrifugation or filtration steps. Residence time distribution (RTD) studies were exploited to achieve the optimal condition of plasmid DNA nanoparticle (pDNA) recovery upon anion exchange adsorbent in this contactor. In addition, the purification experiments were carried out in the expanded bed columns with settle bed height of 6.0 ± 0.2 cm. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-Mr RNA, cDNA and plasmid variants. Subsequently dynamic binding capacity of the adsorbent was calculated while these values decreased with increase in flow velocity. Moreover, the effect of pH upon the performance of this recovery process and the feedstock volume upon the expanded bed anion exchange purification was investigated. The results demonstrated that separation of low-Mr RNA from plasmid DNA isoforms in the range of pH between 5.5 and 7.5 is achievable in this column. The yield of recovery of pDNA in optimal condition was higher than 88.51% which was a superior result in one-pass frontal chromatography. The generic application of simple customized NBG expanded bed column and its potential for the purification and recovery of plasmid DNA as a nanoparticulate bioproduct is strongly discussed.