Modelling the adsorption kinetics of erythromycin onto neutral and anionic resins

In this study the selective adsorption method was chosen to enable the recovery of erythromycin. The following sorbents were tested: neutral resins (XAD-4, XAD-7 and XAD-16) and an anionic resin (IRA-410). A mathematical kinetic model for the adsorption of erythromycin against time, on XAD-4, XAD-7 and XAD-16 resins, is proposed. Both Freundlich and Langmuir models showed a good fit for the sorbents XAD-7 and IRA-410 resins. The highest adsorption efficiency was observed when synthetic neutral resin, XAD-7 and XAD-16, were used. The estimated affinity and concentration factors show that the neutral resins tested are adequate for the selective adsorption of erythromycin. The estimated values of enthalpy and free energy of adsorption, lower than 12 kJ mol^–1 and –2 kJ mol^–1, respectively, indicate that a physiosorption process occurred.     

Improved methods for the extraction of polyphenol oxidase from d'anjou pears

The conditions for extracting polyphenol oxidase (PPO, monophenol monooxygenase, EC 1.14.18.1) from d'Anjou pears have been studied. Water extracts of pear PPO contained artefacts which were present as additional bands on polyacrylamide-gel electrophoresis. Buffer extracts of an acetone powder did not remove sufficient endogenous phenolics to prevent browning of the extract. The following phenolic absorbents, arranged in order of increasing efficiency, reduced the formation of artefacts in extracts of PPO: PVPP, Amberlite XAD-4, Bio-Rad AG 1-X8, and Bio-Rad AG 2-X8. Greatest activity was extracted within a pH range of 5.6–5.9. Anion exchange resins were particularly effective in removing phenolics. XAD-4, AG 1-X8, or AG 2-X8 did not adsorb PPO and reduced the electrophoretically separable bands of PPO activity from 11 in water extracts to 3. The properties of the crude PPO were also studied.     

Wood hydrolyzate treatments for improved fermentation of wood sugars to 2,3-butanediol

Acid-hydrolyzed hardwood contains compounds inhibitory to microorganisms that convert wood sugars to fermentation products such as fuels and chemicals. Several methods of treating acid-hydrolyzed hardwood (hydrolyzate) to reduce the levels of potential microbial inhibitors (acetate, furfural, sulfate, and phenolics) were evaluated. The methods evaluated were precipitation with calcium hydroxide, extraction with organic solvents, treatment with ion-exchange resins, adsorption resins, and activated charcoal. Treatment of the hydrolyzate with an anion exchange resin (Amberlite IRA-400) was the most effective method for removing potential inhibitors. Non-treated hydrolyzate adjusted to pH 6 inhibited growth of a 2,3-butanediol-producing culture of Klebsiella pneumoniae. However, hydrolyzate treated with Amberlite IRA-400 was not inhibitory and resulted in yields of 2,3-butanediol that were greater than 90% of theoretical.     

Adsorbent polystyrene as an aid in plant enzyme isolation

Porous polystyrene (Amberlite XAD-4) adsorbs hydrophobic and surface-active materials from plant extracts and homogenates. With conventional extraction techniques, these materials tend to bind to proteins by hydrophobic interactions. In some cases covalent modification of protein also occurs. Interfering compounds include phenolics, quinones, terpenes and organic isothiocyanates. Polystyrene complements insoluble polyvinylpyrrolidone (PVPP, Polyclar AT), and the combination of these two adsorbents produced superior enzyme extracts from the several plant tissues that were tested. Tested procedures are described, and suggestions are given for adapting the procedures to new plant systems with contaminating natural products varying in quantity and in chemical nature.     

Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.     

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.     

Solvent-impregnated resins as an in situ product recovery tool for phenol recovery from Pseudomonas putida S12TPL fermentations

The sustainable production of fine/bulk chemicals is often hampered by product toxicity and inhibition to the producing micro-organisms. Consequently, the product must be removed from the micro-organisms' environment. To achieve this, so-called solvent-impregnated resins (SIRs) as well as commercial resins have been added to a Pseudomonas putida S12TPL fermentation that produces phenol as a model compound from glucose. The SIRs contained an ionic liquid which extracts phenol effectively. It was observed that the addition of these particles resulted in an increased phenol production of more than a fourfold while the commercial resin (XAD-4) which is widely used in aromatic removal from aqueous phases, only gave a 2.5-fold increase in volumetric production.     

Effect of polymeric adsorbents on the production of sanguinarine by Papaver somniferum cell cultures

The suitability of adsorbent polymeric resins, Amberlite XAD-4 and XAD-7 (Rohm and Hass, Inc.), was investigated for the accumulation of sanguinarine from Papaver somniferum cell cultures. The adsorption and desorption of sanguinarine from aqueous solution was most effective with XAD-7. In addition to sanguinarine, the resins were found to absorb growth regulators and vitamins from the culture medium. Growth inhibition was overcome by delaying for approximately 4 days resin addition after cell inoculation in fresh medium. Resin addition (5% wt/vol) to actively growing uneclicited cultures led to increases in sanguinarine production and release of 30% to 40% and 60%, respectively. The addition of resins to elicited cultures led to increases in alkaloid production of up to 50% to 85% with similar increases in alkaloid release as observed for nonelicited cells. Overall yield of sanguinarine increased from 21 mg . g biomass dry weight(-1) (dw) for elicited cultures to more than 39 mg . gdw(-1) when elicitation was combined with resin addition. Higher quantities of resin (10% to 20% wt/vol) increased marginally the release of sanguinarine into the medium, and on the resin, up to 85% of total production. The use of resin appears promesing for the development of a bioprocess for sanguinarine production by cultured plant cells. (c) 1992 John Wiley & Sons, Inc.     

High-throughput screening of chromatographic separations: IV. Ion-exchange

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.     

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K_p value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425–434, 2017     

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.     

Comparison of affinity tags for protein purification

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.     

Selective adsorption of plant products

The results from this study demonstrate that neutral resins can selectively concentrate specific indole alkaloids from dilute aqueous mixtures. Adsorption was observed to provide a one to two order of magnitude improvement in concentrating these alkaloids, as compared to an equivalent single-staged extraction. Since the sorption correlates with the concentration of the neutral form of the dissolved alkaloid, the adsorption is dependent on both the pH of the medium and the pKa of the alkaloids. Also, adsorption is dependent on specific sorbent-sorbate characteristics. In this study, by exploiting differences in the acid-base properties and the sorption characteristics of specific indole alkaloids, separation factors of 20-30 were observed. Although this behavior is valuable for analytical separations, the present study considers the application to the primary recovery of alkaloids from plant cell processes. Throughout this study it was also observed that the polycarboxylic ester resin (XAD-7) behaved quite differently from the styrene divinylbenzene resin (XAD-4). Despite a lower capacity, the XAD-7 resin was considerably more selective in adsorbing indole alkaloids than the XAD-4 resin. These alkaloids could also be desorbed from the XAD-7 resin by acidifying the liquid, while organic solvents were required to desorb these compounds from the XAD-4 resin.     

Host cell protein adsorption characteristics during protein a chromatography

Protein A chromatography is a critical and ‘gold‐standard’ step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI‐TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back‐bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D‐PAGE was then used to determine individual components associated with resin back‐bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1037–1044, 2012     

Immobilization of glucose isomerase

The immobilization of glucose isomerase (D-xylose ketol isomerase, EC 5.3.1.5) by covalently bonding to various carriers and by adsorption to ion exchange resins was attempted in order to obtain a stable immobilized enzyme which can be used for continuous isomerization of glucose in a column. Of the covalent bonding methods, the colloidal silica-glutaraldehyde method showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Ludox HS-30 bound glucose isomerase column showed a half-life of 24 days and an enzyme usage of 0.07 units per gram of isomerized sugar (d.s, fructose 45%). Of the resins used, the macromolecular type or porous type strongly basic anion exchange resins showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Amberlite IRA-904 resine-bound glucose isomerase showed a half-life of 23 days and an enzyme usage of 0.06 units per gram of isomerized sugar (d.s., fructose 45%). Based on the ease of the immobilization process, the possibility of carrier reuse and the extensive use already achieved by ion exchange resins in the sugar industry, IRA-904 resin was selected as the candidate for commercialization.     

Investigating the combination of single-pass tangential flow filtration and anion exchange chromatography for intensified mAb polishing

There is growing interest within the biopharmaceutical industry to improve manufacturing efficiency through process intensification, with the goal of generating more product in less time with smaller equipment. In monoclonal antibody (mAb) purification, a unit operation that can benefit from intensification is anion exchange (AEX) polishing chromatography. Single-pass tangential flow filtration (SPTFF) technology offers an opportunity for process intensification by reducing intermediate pool volumes and increasing product concentration without recirculation. This study evaluated the performance of an AEX resin, both in terms of host cell protein (HCP) purification and viral clearance, following concentration of a mAb feed using SPTFF. Results show that preconcentration of AEX feed material improved isotherm conditions for HCP binding, resulting in a fourfold increase in resin mAb loading at the target HCP clearance level. Excellent clearance of minute virus of mouse and xenotropic murine virus was maintained at this higher load level. The increased mAb loading enabled by SPTFF preconcentration effectively reduced AEX column volume and buffer requirements, shrinking the overall size of the polishing step. In addition, the suitability of SPTFF for extended processing time operation was demonstrated, indicating that this approach can be implemented for continuous biomanufacturing. The combination of SPTFF concentration and AEX chromatography for an intensified mAb polishing step which improves both manufacturing flexibility and process productivity is supported.     

Improved production of teicoplanin using adsorbent resin in fermentations

AIMS: To use adsorbent resins in fermentations to eliminate toxic effects on growth, reduce feedback repression of production and assist in recovery of teicoplanin. METHODS AND RESULTS: An adsorbent resin was added to the culture broth for the adsorption of teicoplanin. Amberlite XAD-16, Diaion HP-20, charcoal and silica gel were investigated as adsorbent resins. The adsorbed teicoplanin was extracted from the resin by 80% methanol after fermentation. Antibiotic activity was quantified by the disc-agar diffusion assay against Bacillus substilis, and qualitative evaluations were based on HPLC using YMC-Pack ODS-A column. Diaion HP-20 was the most effective adsorbent resin when added at a concentration of 5% (w/v) in the inoculation stage. CONCLUSIONS: Addition of Diaion HP-20 in fermentations eliminated toxic effects on growth and reduced feedback repression of teicoplanin by adsorption. There was a 4.2-fold increase in the quantities of teicoplanin. Addition of adsorbent assisted in the recovery of teicoplanin by reducing the recovery steps. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for the production of teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teicomyceticus. Addition of adsorbent in fermentation increased productivity of teicoplanin by more than five times.     

In situ extraction strategy affects benzophenanthridine alkaloid production fluxes in suspension cultures of Eschscholtzia californica

The effect of contact between cells and extractive phase on secondary metabolite production was investigated in two-phase suspension cultures of Eschscholtzia californica. A system was designed to extract benzophenanthridine alkaloids from the cell culture, without contact between XAD-7 resins and the cells: only medium was recirculated through a column packed with the extractive phase. This strategy was compared to the classic method of addition of resins directly into the cell suspension. Removal of the product directly from the medium enabled important increases in production of alkaloids, namely a 20-fold increase in sanguinarine production and a 10-fold increase in chelerythrine, with high recovery in the resin. The recirculation strategy greatly simplified the production process since the resins are easily recovered from the cell culture and enable harvest of product without termination of culture. However, due to limited flow rate, the recirculation strategy was slightly less effective than direct addition of resins into the cell suspension. In addition to enabling increased production, removal of secondary metabolites from the medium changed metabolic flux distribution, testifying to a complex control mechanism of production.     

Naringinase-catalyzed hydrolysis of naringin adsorbed on macroporous resin

Naringenin, a natural plant flavonoid found in citrus fruits, has been reported to exhibit a wide range of pharmacological functions, including anticancer, antioxidant, antiatherogenic, antithrombotic, and vasodilator activities. Naringenin can be produced from the naringinase (NGase)-catalyzed enzymatic hydrolysis of naringin. However, the poor solubility of naringin in aqueous systems considerably limits the efficiency of naringenin biocatalysis. In this work, a novel substrate adsorption system was proposed for naringin adsorption to increase the efficiency of naringin hydrolysis and naringenin production. Three Amberlite macroporous resins, namely, XAD-4, XAD-7HP and XAD-16, were investigated for their naringin adsorption capacities and effects on NGase hydrolysis. Results indicated that the physical properties of the resins played a critical role in naringin adsorption and naringenin enzymatic synthesis. Naringin hydrolysis was carried out using free and adsorbed substrates. The substrate adsorption strategy could increase the catalytic efficiency at a high naringin concentration. In addition, the reaction conditions for enzymatic naringenin synthesis were optimized, and naringenin was prepared at a liter scale with a high substrate concentration. These results suggested that substrate adsorption is a promising strategy to increase the enzymatic hydrolysis efficiency of naringenin in aqueous systems.