Amplification of bifunctional ligands for calmodulin from a dynamic combinatorial library

A well known strategy to prepare high affinity ligands for a biological receptor is to link together low affinity ligands. DCC (dynamic combinatorial chemistry) was used to select bifunctional protein ligands with high affinity relative to the corresponding monofunctional ligands. Thiol to disulfide linkage generated a small dynamic library of bifunctional ligands in the presence of calmodulin, a protein with two independently mobile domains. The binding constant of the bifunctional ligand (disulfide) most amplified by the presence of calmodulin is nearly two orders of magnitude higher than that of the corresponding monofunctional ligand (thiol).     

蛋白A连续床亲和毛细管色谱柱的制备及性能考察

人免疫球蛋白 G(HIg G)是一种重要的生物大分子 ,是人血浆中的主要成分之一 ,通常采用免疫学的方法测定 .蛋白 A(Protein A)与免疫球蛋白 (HIg G)的 Fc区之间具有很强的特异性亲和作用 ,因而固载蛋白 A的亲和介质可用于免疫球蛋白及单克隆抗体的分离、纯化和分析测定[1～ 3 ] .根据固定相存在形式的不同 ,毛细管色谱柱主要有开管、填充和连续床柱 3种方式 .连续床具有相比高、易制备 (一步合成 )、孔径易控制、不需烧塞子和易改性等优点 .连续床与其它常用的亲和介质 (如球型凝胶颗粒、灌流色谱基质 [4、 5] 、膜介质 [6,7] 等 )相比具…     

Enhanced ligand affinity for receptors in which components of the binding site are independently mobile

Using calmodulin antagonism as a model, it is demonstrated that, under circumstances in which binding sites are motionally independent, it is possible to create bifunctional ligands that bind with significant affinity enhancement over their monofunctional counterparts. Suitable head groups were identified by using a semiquantitative screen of monofunctional tryptophan analogs. Two bifunctional ligands, which contained two copies of the highest-affinity head group tethered by rigid linkers, were synthesized. The bifunctional ligands bound to calmodulin with a stoichiometry of 1:1 and with an affinity enhancement over their monofunctional counterparts; the latter bound with a stoichiometry of 2:1 ligand:protein. A lower limit to the effective concentrations of the domains of calmodulin relative to each other (0.2-2 mM) was determined. A comparable effective concentration was achieved for bifunctional ligands based on higher-affinity naphthalene sulphonamide derivatives.     

Initial evaluation of protein A modified capillary‐channeled polymer fibers for the capture and recovery of immunoglobulin G

A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.     

Mass spectrometry of cis-diamminedichloroplatinum(II) adducts with the dinucleosidemonophosphates d(ApG), d(GpG) and d(TpC) in an ion trap

The detection and fragmentation behaviour of adducts of the chemotherapeutic cis-diamminedichloroplatinum(II) (cisplatin) with the dinucleosidemonophosphates d(ApG), d(GpG) and d(TpC) as model compounds for DNA adducts in an ion trap with electrospray ionization were studied. Mainly the monofunctional adduct, the bifunctional adduct and the bifunctional adduct with platinum bridging two dinucleosidemonophosphates were detected. In addition, several more complex adducts were seen resulting from reactions among these species. Adduct formation was low in the case of d(TpC). Fragmentation could be controlled strongly by varying the temperature of the transfer capillary; furthermore, tandem mass spectrometric (MS/MS) experiments on both the monofunctional and the bifunctional adducts were performed. For the adducts of d(ApG) and d(GpG) losses of NH(3) and HCl were the most dominant reactions, followed by the losses of one, then another two units of 98 amu from the sugar-phosphate backbone, whereas d(TpC)-Pt predominantly forms the dinucleosidemonophosphate. In the gas phase, the conversion of the monofunctional into the bifunctional adducts through binding to another site in the dinucleotide accompanied by loss of NH(3) or HCl could also be observed. The removal of a ligand from the coordination sphere of the square-planar platinum complexes appeared to be the crucial step for the induction of further fragmentation of the dinucleotide ligand. MS(n) experiments of the bifunctional adducts of d(ApG) and d(GpG) revealed different fragmentation pathways involving the loss of phosphoric acid, metaphosphoric acid, deoxyribose units (intact or dehydrated) and the nucleobases in different orders, leaving characteristic binding site-determining fragments. Fragmentation of these ions was also performed, mainly resulting in fragmentation of the bases. The study confirmed the remarkable stability of the platinum-guanine bond compared with other nucleobases.     

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.     

Comparison of protein A affinity sorbents II. Mass transfer properties

Protein A affinity chromatography is the standard purification method for isolation of therapeutic antibodies. Due to improvements in expression technology and optimization of fermentation, culture supernatants with high antibody content must be processed. Recently protein A affinity media with improved adsorption characteristics have been developed. The agarose media MabSelect Xtra and MabSelect SuRe are recent developments of the existing protein A affinity medium MabSelect. MabSelect Xtra is designed to exhibit a higher binding capacity for IgG, and MabSelect SuRe is functionalized with an alkaline stabilized protein A. ProSep-vA Ultra is a porous glass medium with a pore size of 70 nm, also developed to improve the binding capacity. Adsorption was measured in a finite and infinite bath. Mass transfer in these systems could be well described by a model including film and pore diffusion. Mass transfer parameters were used to accurately predict IgG breakthrough in packed bed mode. The dynamic binding capacity of all three media did not change when residence time was at least 4 min. All three media are suited for capture of feed stocks with high antibody content.     

Histidine-epoxy-activated sepharose beads embedded poly (2-hydroxyethyl methacrylate) cryogels for pseudobiospecific adsorption of human immunoglobulin G

The use of highly purified immunoglobulin became among the most powerful adopted strategies in therapeutic trials nowadays. Their role as immunomodulatory and anti-inflammatory agents has widened their scope of use. A novel continuous supermacroporous monolithic cryogels embedded with histidine-epoxy-activated-sepharose beads were synthetized as a new monolithic adsorbents for the separation of immunoglobulin G from human serum. The histidine-epoxy-activated-sepharose beads were embedded into the 2-hydroxyethyl methacrylate (HEMA) cryogels present in frozen aqueous solution inside a plastic syringe. The microstructure morphology of the cryogels was characterized by swelling measurement and scanning electron microscopy. The adsorption of human IgG on the histidine-epoxy-activated-sepharose beads pHEMA cryogels appeared to follow the Langmuir–Freundlich adsorption isotherm model. The maximum IgG adsorption was observed at 4°C and pH 7.4 and was found to be 26.95 mg/g of cryogel which is close to that obtained experimentally (24.49 mg/g). The cryogels were used for several adsorption-desorption cycles without any negligible decrease in their adsorption capacity.     

Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC)

This work aims at understanding the attachment mechanisms and stability of proteins on a chromatography medium to develop more efficient functionalization methodologies, which can be exploited in affinity chromatography. In particular, the study was focused on the understanding of the attachment mechanisms of bovine serum albumin (BSA), used as a ligand model, and protein G on novel amine-modified alumina monoliths as a stationary phase. Protein G was used to develop a column for antibody purification. The results showed that, at lower protein concentrations (i.e., 0.5 to 1.0 mg·mL⁻¹), protein attachment follows a 1st-order kinetics compatible with the presence of covalent binding between the monolith and the protein. At higher protein concentrations (i.e., up to 10 mg·mL⁻¹), the data preferably fit a 2nd-order kinetics. Such a change reflects a different mechanism in the protein attachment which, at higher concentrations, seems to be governed by physical adsorption resulting in a multilayered protein formation, due to the presence of ligand aggregates. The threshold condition for the prevalence of physical adsorption of BSA was found at a concentration higher than 1.0 mg·mL⁻¹. Based on this result, protein concentrations of 0.7 and 1.0 mg·mL⁻¹ were used for the functionalization of monoliths with protein G, allowing a maximum attachment of 1.43 mg of protein G/g of monolith. This column was then used for IgG binding–elution experiments, which resulted in an antibody attachment of 73.5% and, subsequently, elution of 86%, in acidic conditions. This proved the potential of the amine-functionalized monoliths for application in affinity chromatography.     

KINETIC ANALYSIS OF THE PHOTOREACTION (365 nm) BETWEEN PSORALEN AND DNA

Abstract— Psoralen complexed with DNA under irradiation at 365 nm forms monofunctional and bifunctional adducts with pyrimidine bases of DNA. while the fraction of psoralen which remains free in the aqueous solution forms photodimer and photooxidation products. We havc studied the photoreaction betwecn psoralen and DNA from a kinetic point of view. Only some products of this photoreaction could be measured quantitatively. Thus. in order to present a picture of the overall photoreaction. we have represented this by a mathematical model consisting of a system of seven differential equations. Utilizing all the experimental data that it was possible to obtain. we were able to estimate rate constants for formation of all photoproducts. From the general picture thus obtained. we conclude that monofunctional adducts are formed in a four to one ratio to bifunctional adducts. Among the monofunctional photoproducts. 3,4-cycloadducts occur with a higher yield than 4',5'-cycloadducts. The combined rate constant of formation of photodimer and photooxidation products of psoralen is small. indicating that they are marginal by-products relative to the cycloadducts formed in the photoreactions with DNA.     

Screening and chromatographic assessing of a novel IgG biomimetic ligand

A novel biomimetic ligand, N-benzyloxycarbonyl-l-tyrosine (N-cbz-l-Tyr), was screened by a combination method of molecular docking and immobilized receptor technique. Then, N-cbz-l-Tyr was immobilized on Sepharose CL-4B to prepare a specific affinity adsorbent for immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on the adsorbent gave an association constant (K(a)) of 4.91 x 10(6) m(-1) and a theoretical maximum adsorption capacity of 17.3 mg IgG/mL gel. IgG with a purity of 98% was separated from human plasma by this new affinity adsorbent.     

小鼠血清IgG的蛋白G灌注亲和色谱行为的研究

通过实验考察了小鼠血清ＩｇＧ的蛋白Ｇ灌注亲和色谱行为,虽然未发现非特异性吸附,但在通常情况下对ＩｇＧ存在不可逆性吸附。洗脱液组成及ｐＨ的不同决定ＩｇＧ在灌注亲和色谱柱上具有不同的解离常数ＫＤ,产生不同的色谱行为。流动相的流速对ＩｇＧ与蛋白Ｇ的结合／解离动力学过程产生一定的影响。结果表明,对于小鼠血清中的ＩｇＧ灌注亲和色谱的分离,由于解离速率影响到传质过程,使之在高流速下的分离受到一定的限制。     

ProteinA亲合膜色谱柱的性能及从人血浆中纯化免疫球蛋白的研究

以木纤维为基质,与甲基丙烯酸环氧丙酯共聚接枝合成了复合膜介质,用复合膜介质制备了ｐｒｏｔｅｉｎＡ亲合膜色谱柱,考察了ｐｒｏｔｅｉｎＡ亲合膜色谱柱液相流动特性和吸附性能。实验证明：流速与亲合膜色谱柱柱压呈线性关系,当流速为３ｍＬ／ｍｉｎ时,柱压为１６０ｋＰａ。免疫球蛋白（ＩｇＧ）浓度和上样速度是影响ｐｒｏｔｅｉｎＡ键合容量的重要因素,对其进行了优化研究。用动态吸附法确定了对人ＩｇＧ动态最大吸附能力可达２１．７ｍｇ／ｇ（干介质）。     

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.     

Stereospecificity and enantioselectivity in the binding of the platinum(II) complex [PtCl2(tmdz)] (tmdz = 5,5,7-trimethyl-1,4-diazacycloheptane) to dinucleotides and oligonucleotides

The two stereoisomers formed on reaction of each of the enantiomers of [PtCl2(tmdz)] with d(GpG) have been identified by using one- and two-dimensional 1H NMR spectroscopy. For both isomers formed with the R enantiomer the 3'-H8 shifts are downfield from those for the 5'-H8. For the S enantiomer the reverse is observed, showing that the bulky tmdz ligand determines the pattern of shifts. Models of these isomers generated by molecular mechanics show that the bulky tmdz ligand limits the rotation of the guanine bases and enforces right-handed (R2) canting for both isomers formed by the R enantiomer and left-handed (L1) canting for those formed by the S enantiomer. The pattern of H8 shifts is the opposite to that expected for these cantings; this suggests that other factors may play a role in determining these shifts. The interactions between the tmdz and d(GpG) ligands are also shown by molecular mechanics and the broadness of the H8 NMR signals to influence the tendency of the coordinated guanine bases to rotate about their Pt-N7 bonds. Reaction of each of the enantiomers with a 52 base-pair nucleotide, with a total of six GpG binding sites, resulted in the formation of only one of the stereoisomers in each case, the first reported case of complete stereoselectivity, or stereospecificity, in the reaction of Pt complexes with DNA. The observed stereoisomers were identified by comparison with the properties of the d(GpG) complexes. Molecular mechanics models of the adducts with duplex DNA show that the nonformation of one stereoisomer is consistent with the steric bulk of the tmdz ligand preventing closure from the monofunctional adduct to the bifunctional adduct. Enantioselectivity is also observed in that the R enantiomer forms more monofunctional adducts than bifunctional (59:41), whereas the S enantiomer forms more bifunctional adducts (27:73). The origins of this enantioselectivity must be at the level of monofunctional adduct formation and this has been investigated by molecular mechanics modelling.     

Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces. Binding isotherms for both interactions were measured by different physiochemical techniques: surface plasmon resonance, fluorescence spectroscopy and dynamic light scattering. Both qualitatively and quantitatively there is little difference in binding affinities between the bifunctional aptamers and their monofunctional components. The respective K_d values for streptavidin binding in the monofunctional aptamer and in the theophylline bifunctional aptamer were 12 nM and 65 nM, respectively whilst the K_d values for theophylline binding in the monofunctional aptamer and the streptavidin bifunctional aptamer were 300 nM and 120 nM. These results are consistent with treating each aptamer sequence as a module that can be combined with others without significant loss of function. This allows for the use of streptavidin based immobilization strategies without either the cost of biotinylated dNTPs or the variable yields associated with the chemical biotinylation of RNA.     

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.     

Gold Nanoparticle-based “Mix and Measure” Fluorimetric Assays to Quantify Antibody Titer

Monoclonal antibodies (mAbs) for treatment of human diseases are typically human or humanized Immunoglobulin G (IgG) produced in mammalian cell lines. A rapid, less tedious, and high throughput method to quantify mAbs is in demand to accelerate mAb production efficiency. To quantify mAb titer, we developed gold nanoparticle (AuNPs)-based “mix and measure” fluorimetric assays by exploiting AuNPs’ fluorescence quenching ability. The AuNPs are functionalized by an Fc binding protein, i. e. protein G, which binds human IgG and fluorescently labeled rat IgG (Alexa Fluor 488-rat IgG) with differential affinity. The assays can be in competition or displacement format. The competitive binding of human IgG drug and the labelled rat IgG to protein G-coated AuNP lead to varied fluorescent intensity that is proportional to the amount of human IgG analte; or the displacement of the labelled rat IgG from protein G-coated AuNP by human IgG can lead to fluorescent recovery that is also proportionally related to human IgG concentration. The assays can quantify therapeutic mAbs in the range of 10–1,000 mg/L, demonstrated for Herceptin, Avastin, and Humira in cell culture media. The assays have fast turn over time (within 15 min). They can be performed in microplates and are suitable for high throughput “on-line” or “at-line” measurement in mAbs production lines.