Enantioselective addition of phenylacetylene to aldehydes catalyzed by polymer‐supported titanium(IV) complexes of β‐hydroxy amides

A series of polymer‐supported chiral β‐hydroxy amides and C₂‐symmetric β‐hydroxy amides have been synthesized and successfully used for the enantioselective addition of phenylacetylene to aldehydes. High yields (up to 93%) and enantioselectivities (up to 92% ee) were achieved by using polymer‐supported chiral β‐hydroxy amide 4b . The resin 4b is reused four times, giving the product with enantioselectivity 80% ee. Fortunately, it is found that this heterogonous system is suitable not only for aromatic aldehydes but also aliphatic aldehyde. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Role of Swelling Pressure on the Distribution Coefficient of Maltooligosaccharide in a Cation-exchange Resin

The distribution coefficients of maltooligosaccharides with a degree of polymerization of 1 to 7 in cation-exchange resins (Amberlites HFS-471X and CG-60) with various crosslinkages and of counterion forms were used to estimate the swelling pressure of the resins. The distribution coefficient of glucose in the resin with 6 % divinylbenzene of various ionic forms was measured by varying the ionic strength of the eluent. Although the resins shrank with increasing ionic strength, the distribution coefficient became larger. These results suggest that the swelling pressure of an ion-exchange resin plays an important role in the distribution of a non-electrolyte such as maltooligosaccharide to the resin. A simple method to estimate the swelling pressure is also described.     

Comparative evaluation of four trityl-type amidomethyl polystyrene resins in Fmoc solid phase peptide synthesis.

Four trityl-type (i.e. non-substituted trityl-, o-Cl-trityl-, o-F-trityl- and p-CN-trityl-) amidomethyl polystyrene resins were evaluated comparatively, in terms of the stability of the trityl-ester bond in slightly acidic dichloromethane solutions, and the p-CN-trityl-amidomethyl polystyrene resin was found to be the most stable of them. The above resins were applied, in parallel with Wang benzyl-type resin, well known for its stability in mild acidic conditions, to the Fmoc solid phase synthesis of the 43-amino acid residue long bioactive peptide thymosin beta-4. Independent of their differences in acid sensitivity, the resins seemed to function equally well under the conditions used, since pure thymosin beta-4 was obtained with a final yield of approximately 30% from each resin. The trityl-type amidomethyl polystyrene resins were also applied, in parallel with the Wang resin, to the Fmoc solid phase synthesis of a bioactive peptide containing proline at its C-terminus, i.e. the N-terminal tetrapeptide of thymosin beta-4, AcSDKP. In this case, the best yield (87%) was obtained with the o-Cl-trityl-amidomethyl polystyrene resin, which may be the resin of choice, of those studied, for the Fmoc solid phase peptide synthesis.     

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.     

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.     

Preparation of high purity biphenyl cyclooctene lignans from Schisandra extract by ion exchange resin catalytic transformation combined with macroporous resin separation

In this study, ester-bond biphenyl cyclooctene lignans were efficiently hydrolytically degraded into free biphenyl cyclooctene lignans by ion exchange resin transformation and simultaneous removal of impurities by macroporous resin. The OH-type strongly basic anion exchange resin 201×7 was the best one, and the dynamic hydrolysis efficiency was 146.7±5.0%. HPD5000 macroporous resin, which offered higher adsorption and desorption capacities and faster adsorption than other resins. The purity of free biphenyl cyclooctene lignans in the product increased from 5.14±0.24% to 79.67±0.0.67%. After dynamic catalytic transformation by 201×7 resin combined with purification of HPD5000 resin, the yield and the purity of free biphenyl cyclooctene lignans in the product were 132.1±4.7% and 80.91±3.53%, respectively.     

Soft shell resins for solid-phase peptide synthesis.

Soft shell (SS) resins with a lightly or noncross-linked shell layer were prepared by reducing the amount of cross-linking agent, divinylbenzene (DVB), during seed suspension polymerization from polystyrene (PS) resin. These SS resins have a lower swelling volume than that produced by normal cross-linking. Despite its lower swelling, however, SS (10-00) resin, which consists of the 1% DVB-cross-linked core and the noncross-linked surface layer, showed higher efficiency in peptide synthesis compared with 1% DVB-PS resin and other SS resins.     

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.     

大孔树脂提取链霉菌4903菌株除草活性物质的研究

化感活性物质的提取方法是研究化感作用的重要步骤,对化感链霉菌4903的前期研究证明其具有抑菌及除草活性。本文选用了4种大孔吸附树脂(X-5, NKA-II, AB-8和HP-20)对化感链霉菌4903发酵液的除草活性物质进行提取。实验结果显示:树脂AB-8对链霉菌4903菌株发酵液除草活性成分具有较好的吸附性能和较易被解吸的特性,树脂AB-8对链霉菌4903菌株发酵液的静态吸附量约可达到树脂体积的7倍。用树脂2-3倍体积的80%乙醇溶液则可以把吸附的除草活性物质全部从树脂中解吸出来。AB-8适合用于链霉菌4903菌株发酵液除草活性物质的提取和纯化。     

Affinity Chromatography: An Enabling Technology for Large‐Scale Bioprocessing

Affinity chromatography (AC) has been used in large‐scale bioprocessing for almost 40 years and is considered the preferred method for primary capture in downstream processing of various types of biopharmaceuticals. The objective of this mini‐review is to provide an overview of a) the history of bioprocess AC, b) the current state of platform processes based on affinity capture steps, c) the maturing field of custom developed bioprocess affinity resins, d) the advantages of affinity capture‐based downstream processing in comparison to other forms of chromatography, and e) the future direction for bioprocess scale AC. The use of AC can result in economic advantages by enabling the standardization of process development and the manufacturing processes and the use of continuous operations in flexible multiproduct production suites. These concepts are discussed from a growing field of custom affinity bioprocess resin perspective. The custom affinity resins not only address the need for a capture resin for non‐platformable processes, but also can be employed in polishing applications, where they are used to define and control drug substance composition by separating specific product variants from the desired product form.     

Comparison of procedures for directly obtaining protected peptide acids from peptide-resins.

The preparation of small-sized protected peptide acids related to cholecystokinin and gomesin was attempted using peptide-Kaiser oxime resins (KOR) as starting materials. For comparison, peptide-2-Cl-trityl resin (CLTR) was also employed. While peptide detachment from KOR was achieved through the oxime ester bond hydrolysis mediated by DBU, hydroxide ion or Ca(+2) ion, peptide release from CLTR was accomplished through acid-catalysed hydrolysis of the peptide-resin ester linkage. Amino acid analysis of the peptide-resins before and after peptide release allowed the calculation of the reaction yields. RP-HPLC and LC/ESI-MS of the resulting crude peptides allowed estimation of their quality. The data collected indicated that: (i) among the procedures used for peptide displacement from KOR, the one employing DBU was the most efficient since it furnished all model protected peptide acids with the highest quality in a very short time; (ii) although slow, Ca(+2)-assisted peptide detachment from KOR was selective and was suitable for generating high-quality protected peptide acids containing up to five residues; (iii) even though the protocols employed for peptide release from CLTR have shown to be appropriate, the one employing 1% TFA/DCM was the most productive; (iv) in terms of product quality, DBU-catalysed peptide detachment from KOR was similar to TFA-catalysed peptide release from CLTR although the latter was more productive. These findings are relevant to peptide chemists in general, but especially to those interested in preparing acyl donors for convergent peptide syntheses by the t-Boc chemistry.     

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.     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.     

Using high throughput screening to define virus clearance by chromatography resins

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process‐related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non‐infectious retrovirus‐like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96‐well batch‐binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow? (QSFF) and Capto adhere?, a mixed mode resin. QSFF batch‐binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product‐specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96‐well batch‐binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins. Biotechnol. Bioeng. 2013; 110: 1984–1994. © 2013 Wiley Periodicals, Inc.     

Viral clearance studies on new and used chromatography resins: critical review of a large dataset.

Viral clearance studies for na?ve and maximally cycled chromatographic resins used for cGMP recombinant protein production are reviewed for three products, comprising 10 different chromatographic steps, including affinity, ion exchange, immobilized metal ion affinity, and hydrophobic interaction modes. Thirty-two separate studies were conducted (over 90 runs in total). No consistent reductions in model virus clearance were observed with used resins. The results address the reproducibility of virus clearance studies conducted by different scientists over several years at multiple contract labs. The log reduction values (LRVs) are typically within 0.5 LRVs for new and used resin, but varied as much as 2 LRVs for resins showing no functional deterioration. This relatively large difference is not believed to reflect resin changes, but highlights the challenges encountered in modeling column clearance. Production column performance and cleaning efficacy are demonstrated for these steps by trending mock runs, impurity removal and product recovery. No deterioration in cGMP column performance is seen over the established resin lifetimes, confirming that the resin regeneration and sanitization procedures restore the resins to a suitable initial state without damage. It is proposed that for some chromatography steps, the combination of lab-scale cycling studies confirming consistent performance throughout the resin lifetime and monitoring of cGMP manufacturing preclude the need for virus clearance studies on maximally cycled resin.     

Introducing Biobased Materials into the Electronics Industry

Abstract: Lignin, a biopolymer formed in the cell walls of plants, is a by-product of paper manufacturing. In research at IBM, it was incorporated into a resin used in the fabrication of printed wiring boards (PWB) for the microelectronics industry. The resin had physical and electrical properties similar to those of current laminate resins. PWBs fabricated from the lignin-based resin passed most of the standard physical, electrical, and reliability tests for an "FR4"-grade laminate. A comparison of the lignin-based resin and current resins via life-cycle assessment indicated up to 40% lower energy consumption for the biobased resin. Large-scale manufacture of lignin-based resins would require an inexpensive source of lignin with low ionic contamination.     

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.     

Evidence for an angiotensin‐(1‐7) neuropeptidase expressed in the brain medulla and CSF of sheep

Angiotensin‐(1‐7) [Ang‐(1‐7)] is an alternative product of the brain renin‐angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang‐(1‐7) to the inactive metabolite product Ang‐(1‐4) in CSF of adult sheep. This study purified the peptidase 1445‐fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o‐phenanthroline and EDTA, as well as the mercury compound p‐chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin‐converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV‐390 was a potent inhibitor of Ang‐(1‐7) hydrolysis (Ki = 0.8 nM). Kinetic studies using ¹²⁵I‐labeled Ang‐(1‐7), Ang II, and Ang I revealed comparable apparent K_m values (2.6, 2.8, and 4.3 μM, respectively), but a higher apparent V_max for Ang‐(1‐7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang‐(1‐7) to Ang‐(1‐4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin‐13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang‐(1‐7) within the brain.

     

Separation of scutellarin from crude extracts of Erigeron breviscapus (vant.) Hand. Mazz. by macroporous resins

Scutellarin, a flavone glycoside, popularly used in the treatment of heart disease, has been efficiently separated using macroporous resins from crude extracts of Chinese medicinal plant Erigeron breviscapus (vant.) Hand. Mazz. HPD-800 resin offered the best adsorption and desorption capacity for scutellarin among the eight macroporous resins tested, and its adsorption data at 25 degrees C fit best to the Langmuir isotherm. The dynamic adsorption and desorption experiments have been carried out on a HPD-800 resin packed column to optimize the separation process of scutellarin from the crude extracts of E. breviscapus. After one run treatment with HPD-800 resin, the scutellarin content in the product was increased 15.69-fold from 2.61% to 40.96% with a recovery yield of 95.01%. The preparative separation process via adsorption-desorption method developed in this study provides a new approach for scale-up separation and purification of scutellarin for its wide pharmaceutical use.     

Effect of flask closure method and post‐pressing time on the upper denture base adaptation

doi:10.1111/j.1741‐2358.2009.00307.x
Effect of flask closure method and post‐pressing time on the upper denture base adaptation Objectives: The purpose of this study was to evaluate the effect of flask‐closure methods, post‐pressing times and acrylic resins on denture base adaptation. Materials and methods: The resins were flasked using a hydraulic press and closed with the traditional clamp or RS system. Conventional heat‐cure resin was polymerised immediately or at 6 h post‐pressing at 74°C for 9 h. Rapid cycle heat‐cure resin was polymerised in boiling water for 20 min. After cooling, the bases were deflasked and the sets of cast‐base transversally sectioned in the regions distal to the canine, mesial to the first molar and in the posterior palatal zone. The adaptation was measured with an optical microscope (0.0005 mm) at five reference points for each section. Data were analysed using anova and Tukey’s test (α = 0.05). Results: Traditional clamp and immediate post‐pressing time improved base adaptation for conventional heat‐cure resin. Both post‐pressing times showed most accurate base adaptation for conventional heat‐cure resin when the traditional clamp was used. Immediate post‐pressing time improved base adaptation for conventional heat‐cure resin and the 6‐h delay in time was significant for the rapid cycle heat‐cure resin. Conclusions: Traditional clamp and immediate post‐pressing time improved base adaptation for conventional heat‐cure resin.