Effect of histidine residues in antigenic sites on pH dependence of immuno-adsorption equilibrium

Summary Polyclonal anti-myoglobin antibodies were fractionated into five subpopulations directed against five specific antigenic sites, respectively. The equilibrium characteristics of each subpopulation and orginal anti-myoglobin immobilized to CNBr-activated Sepharose 4B were compared. The four subpopulations of antibodies lost their binding abilities at around pH 4.5 because of the conformational changes of myoglobin. However, the subpopulation directed against the region containing three histidine residues dissociated with the antigenic site at higher pH, and such equilibrium characteristics were considered to be caused by the dissociation characteristics of histidine residues. Therefore, the effects of histidine modification in BSA on the adsorption capacities of original anti-BSA antibody and a pH sensitive fraction of it were compared. The adsorption capacity of the pH sensitive fraction showed greater decrease than that of original antibody by histidine modification in BSA. These results imply that the antigenic sites in which histidine residues play an important role for the binding to antibodies show equilibrium characteristics sensitive to pH.     

An evaluation of elution techniques in the study of immune complex glomerulonephritis.

Antigen and antibody from glomerular immune complex deposits in rabbits with experimental bovine serum albumin-(BSA) induced chronic serum sickness (CSS) were quantitated in elutes from kidneys in which a portion of the antigen and antibody had been radiolabeled. The largest quantities of 125I BSA eluted with 1 M roprionic acid at pH 2.7 (86%) and 0.1 M borate buffer at pH 11.25 (80%). However, these buffers yielded less functional anti-BSA antibody than 0.02 M citrate buffer at pH 3.2 (344 mug/g kidney). Citrate buffer-eluted anti-BSA antibody was reactive in immunodiffusion, immunofluorescence, and radiolabeled BSA binding test systems, but complement fixation was impaired relative to chaotropic ion-eluted antibody. It was found that up to 75% of the eluted antibody was lost to further study by recombination with eluted BSA. This could be prevented by fractionation of the dissociated eluate before neutralization. IgG fractionated eluates were successfully fluorescein conjugated or radiolabeled for use as reagents. Elution of cryostat sections of CSS kidney was also studied; BSA, IgG, and complement (C3) eluted in parallel, and sub-microgram quantities of anti-BSA antibody were recovered.     

Assessment of the use of cyanogen bromide-activated Sepharose in analytical and preparative immunoadsorption

Procedures are described for the analytical and preparative purification of antigens based on their specific interaction with their complementary antibody immunoadsorbents prepared from cyanogen bromide-derivatized macroporous agarose matrices. In principle, the antigen to be purified in the affinity chromatography/immunoadsorption process should bind specifically and reversibly to the attached antibody, while other proteins pass through unretarded. In the case of tight binding, elution of the antigen is achieved by the use of eluting solutions of very high or very low pH, or with the use of chaotropic solutions such as 3 m KSCN. The performance of immunoadsorbents prepared from Sepharose 4B have been studied with the aim of improving the efficient utilization of immunoadsorption techniques. As a model, human serum was applied serially to several columns of Sepharose 4B sheep anti-human IgG which were then subjected to a number of successive adsorption/desorption cycles. Loading the columns with increasing amounts of serum showed that the performance was best when the antigen load was approximately threefold the ideal binding capacity. By limiting the amount of immobilized protein and carefully controlling the antigen load, significant improvements in yield and purity have been achieved. Antigen loads of threefold the potential binding capacity of the immunoadsorbent column results in the optimal yield of antigen with high purity and significant concomitant reduction in non-specific interference from other serum proteins. The non-specific adsorption which is an inherent problem and which leads to considerable inactivation of the covalently coupled antibody is highlighted. Although the popularity of such matrices is probably unsurpassed, it is clear that use has been made of them very frequently without an examination of quantitative aspects or side reactions.     

Studies on adsorption isotherms of endotoxin and BSA using an affinity column

In this paper, an adsorbent with dimethylamine ligand for endotoxin removal was prepared and used to study the adsorption isotherms of endotoxin and BSA. The results showed that as the introducing of endotoxin, the maximum adsorption capacity q_m of BSA increased from 7.24 to 7.74 mg/mL and the apparent association constant K_A of BSA decreased from 14.06 to 11.48 mL/mg. The adsorption isotherms of BSA changed from Langmuir model to Multilayer model. All these gave the direct evidences to construct the adsorption process. By comparing adsorption isotherms of endotoxin with BSA, it was found that the apparent association constant K_A of endotoxin was much higher than that of BSA.     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.     

Application of dye-ligands affinity adsorbent in capturing of rabbit immunoglobulin G

The applicability of dye-ligands attached to an expanded bed chromatography quartz base matrix (Streamline™) for the affinity bioseparation of rabbit immunoglobulin G (IgG) was investigated. Reactive Green 5 (RG-5) immobilized onto adsorbent was selected for capturing of rabbit-IgG due to its higher binding capacity compared to other dye-ligands possessing similar ligand density. Adsorption parameters such as pH, temperature, ionic strength and initial rabbit-IgG concentration were optimized for the adsorption of rabbit-IgG on the RG-5-immobilized adsorbent. The highest rabbit-IgG adsorption was recorded in pH 7.0, while the maximum binding capacity for BSA was achieved at pH 4.0. The adsorption of rabbit-IgG on RG-5-immobilized adsorbent was declined as the increase of ionic strength. There is no significant influence of temperature against adsorption efficiency of RG-5-immobilized adsorbent for rabbit-IgG. The adsorption phenomenon of rabbit-IgG on RG-5-immobilized adsorbent appeared to follow the Langmuir–Freundlich adsorption isotherm model. The theoretically maximum binding capacity (q_m) of RG-5-immobilized adsorbent estimated from this isotherm was 49.3 mg ml⁻¹, which is very close to that obtained experimentally (49.0 mg ml⁻¹). About 50% of bound BSA on RG-5-immobilized adsorbent in binary adsorption system was removed with washing buffer containing 1 M NaCl.     

Adsorption and elution behaviors of bovine serum albumin in metal-chelated affinity cryogel beds

Metal-chelated supermacroporous cryogels are effective supports for affinity chromatographic separation of biomolecules in downstream processes. In this work, polyacrylamide cryogel beds were prepared in glass columns (16 mm inner diameter) and coupled with iminodiacetic acid (IDA). These cryogels were loaded with Zn²⁺ and Ni²⁺ and the so-called Zn²⁺-IDA-cryogels and Ni²⁺-IDA-cryogels were obtained. Permeabilities and height equivalent to theoretical plates (HETPs) of these cryogel beds were measured and the cryogel structure was analyzed using scanning electron microscopy (SEM). Bovine serum albumin (BSA) was employed as a model protein to elucidate the adsorption and elution behaviors of these cryogels under various conditions, such as different flow rate, solution pH, and composition of the eluents. The results showed that the Zn²⁺-IDA-cryogels and Ni²⁺-IDA-cryogels in this study had interconnected supermacropores and high water permeabilities (∼10⁻¹¹ m²). The loading flow velocity had a weak influence on the breakthrough curves and binding capacities for BSA, while the solution pH had an evident effect on the binding capacities for BSA in these cryogels. Maximum binding capacity for BSA was observed near the isoelectric point of BSA. The bound BSA can be eluted effectively using an imidazole solution. A low-eluting flow rate was found to be beneficial to the elution process. Possible mechanisms were proposed and discussed.     

The influence of bakers’ yeast cells on protein adsorption performance in dye-ligand expanded bed chromatography

The influence of whole yeast cells (0–15% w/v) on the protein adsorption performance in dye-ligand chromatography was explored. The adsorption of a model protein, bovine serum albumin (BSA), was selected to demonstrate this approach. The UpFront adsorbent (ρ=1.5 g/cm³) derivatised with Cibacron Blue 3GA and a commercially available expanded bed column (20 mm i.d.) from UpFront Chromatography, Denmark, were employed in the batch binding and expanded bed operation. The BSA binding capacity was demonstrated to not be adversely affected by the presence of yeast cells. The dynamic binding capacity of BSA at aC/C ₀=0..1 biomass concentration of 5, 10, 15% w/v were 9, 8, and 7.5 mg/mL of settled adsorbent, respectively.     

Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples

The increasing concern about ochratoxin A (OTA) contamination of different food and feedstuffs demands high-performing detection techniques for quality assessment. Two indirect competitive enzyme-linked immunosorbent assay (ELISA) strategies were investigated for the development of OTA electrochemical immunosensors based on different OTA immobilisation procedures. Immunosensors based on avidin/biotin-OTA showed enhanced performance characteristics compared to those based on the adsorption of bovine serum albumin (BSA)-OTA conjugate. Performance of polyclonal (PAb) and monoclonal (MAb) antibodies against OTA was compared, showing at least one-order of magnitude lower IC(50) values when working with MAb. Alkaline phosphatase (ALP)- and horseradish peroxidase (HRP)-labelled secondary antibodies were evaluated. Both conjugates led to similar results when working with OTA standard solutions in buffer. However, whereas electroactive interferences present in spiked wine samples did not affect HRP-labelled immunosensors (4% slope deviation), they were likely oxidised at 0.225 V versus Ag/AgCl, the working potential for ALP-labelled immunosensors (25% slope deviation). Considering 80% of antibody binding as the limit of detection, values of 0.7 and 0.3 ng/mL for HRP- and ALP-labelled immunosensors respectively, validate these immunosensors as useful screening tools to assess OTA levels in wine.     

Agar-based magnetic affinity support for protein adsorption

Magnetic colloidal particles were prepared by a coprecipitation method. The particles were composed of nanometer-sized superparamagnetic Fe(3)O(4) particles stabilized by lauric acid. Then, magnetic agar gel beads were produced by a water-in-oil emulsification method using a mixture of agar solution and the magnetic colloidal particles as the aqueous phase. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare an agar-based magnetic affinity support (MAS) for protein adsorption. The support showed good magnetic responsiveness in a magnetic field. Bovine serum albumin (BSA) was used as a model protein to test adsorption equilibrium and kinetic behavior of the MAS. The adsorption equilibrium of BSA to the MAS was described by the Langmuir-type isotherm. Adsorption capacity of the MAS for BSA was up to 25 mg/mL at a CB coupling density of 1.6 micromol/mL. The effect of ionic strength on BSA adsorption was complex, exhibiting a maximum capacity at an ionic strength of 0.06 mol/L. The adsorption of BSA to the MAS was also influenced by pH. Uptake rate of BSA to the MAS was analyzed using a pore diffusion model. The pore diffusion coefficient was estimated to be 1.75 x 10(-11) m(2)/s. Finally, recycled use of the MAS demonstrated the stability of the MAS in protein adsorption and magnetic responsiveness.     

Reusability of avidin-biotinylated immunoglobulin Y columns in immunoaffinity chromatography

Reusability of avidin-biotinylated IgY columns for immunoaffinity chromatography was examined by repeated use and regeneration. An enzyme-linked immunosorbent assay-elution assay using CovaLink NH microtiter plates was used to find the optimal conditions for regeneration of columns. Actigel avidin-biotinylated IgY column retained about 90% of its initial IgG binding capacity after 50 cycles, with 0.1 M glycine-HCl buffer, pH 2.8, as eluent, requiring no regeneration. However, IgG binding capacity of UltraLink avidin-biotinylated IgY column gradually decreased to 75 and 65% after 10 and 20 cycles, respectively, with the commercial eluent, Actisep. Results from the CovaLink NH system agreed with those from UltraLink avidin-biotinylated IgY columns. The UltraLink avidin-biotinylated IgY column was regenerated twice, by applying 8 M guanidine-HCl, pH 1.6, to dissociate biotinylated IgY antibodies from the column. About 40 and 25% of IgG binding capacities remained after the first and second regeneration. By applying new biotinylated IgY to the treated columns, about 95 and 90% of the initial IgG binding capacity before any treatment were recovered. These results demonstrated that avidin-biotinylated IgY columns are reusable with or without regeneration depending on the avidin-immobilized matrix.     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

Enhancing protein capacity of rigid macroporous polymeric adsorbent.

A macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) resin was synthesized and modified with diethylamine to yield an anion-exchange resin suitable for protein adsorption. Efforts were made to enhance protein ion exchange capacity of the resin by investigating the copolymer composition. Different synthesis recipes were attempted, and the resultant resins were characterized by measuring the specific surface area and the adsorption ability using bovine serum albumin (BSA) as a model protein. The intraparticle pore size distribution measured by mercury porosimetry showed that the pores in the range of 40-120 nm took 75% of the total pore volume, indicating that the ion exchanger was favorable for protein adsorption. BSA capacity obtained with an appropriate recipe was as high as 78.6 mg/g wet resin or 50 mg/mL packed volume, which was higher than the capacities of some commercially available ion exchangers. Moreover, by using a pore diffusion model, the effective pore diffusivity of BSA was found to be 5.5 x 10(-12) m(2)/s, similar to those in the commercial ion exchangers.     

Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer

Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.     

LiCl-induced improvement of multilayer nanofibrous lipase for biodiesel synthesis

A unique method that applied a multilayer-immobilization strategy was developed to prepare nanofibrous enzymes for biosynthesis. LiCl co-electrospun with polyurethane nanofibers enabled strong physical adsorption of bovine serum albumin (BSA), forming the first layer of protein on the nanofibers; lipase AK was subsequently crosslinked to BSA as an outer layer of enzyme. The content of LiCl in nanofibers was found to be a sensitive factor affecting the activity and stability of the immobilized lipase. For biodiesel synthesis from soybean oil and methanol in isooctane, the reaction rate catalyzed by nanofibrious lipase carrying 5 wt% LiCl was 6.6-fold higher than fibers without LiCl, with a conversion of 91% was achieved within 2 h. LiCl also induced much improved enzyme stability. The nanofibrous lipase with 5% LiCl could be repeatedly used for 42 cycles without apparent activity loss, while the immobilized lipase without LiCl lost over 90% activity within 13 reuse cycles.     

Expanded and packed bed albumin adsorption on fluoride modified zirconia

The expanded bed characteristics of 75-103microm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2x expanded bed, and a 3x expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 +/- 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 micrometer) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times ( approximately 0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion.Copyright 1998 John Wiley & Sons, Inc. Bioeng 60: 333-340, 1998.     

Applicability of airlift draft-tube fluidized bioreactors for binary protein mixture bioseparation

An airlift draft-tube fluidized bioreactor has been designed and tested for applications in protein bioseparation. Operating parameters and geometrical dimensions of the bioreactor were optimized to ensure fluid circulation in a defined cyclic pattern between the riser and the downcomer. The overall directionality of liquid flow generates homogeneous field of low shear and achieves good mixing efficiency. Bioseparation of proteins was achieved from solutions containing both BSA and BHb at different initial concentrations and at pH 7. Similar adsorption capacities of both proteins were observed in single protein adsorption experiments at pH 7. Compressibility of BHb allowed for high adsorption capacity, in addition to the hydrophobic interaction forces. Apparently the homogeneous and lower shear generated by the airlift bioreactor reduces the compressibility of adsorbed BHb. This allowed for higher BSA adsorption from solutions containing BSA and BHb mixtures. Conventional batch adsorption experiments showed more adsorption of BHb, which reduces bioseparation efficiency.     

Ion exchange chromatography of antibody fragments

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.     

Biosorption of Cr (VI) from aqueous solution by Rhizopus nigricans

The study was aimed to quantify the Cr sorption ability of powdered biomass of Rhizopus nigricans at the best operating conditions. The influence of solution pH, agitation, Cr (VI) concentration, biomass dosage, contact time, biomass particle size and temperature were studied. The optimum pH for biosorption of Cr (VI) was found to be 2.0. Higher adsorption percentage was noted at lower initial concentrations of Cr ions, while the adsorption capacity of the biomass increased with increasing concentration of ions. Optimum biomass dosage was observed as 0.5% (w/v). More than 75% of the ions were removed within 30 min of contact and maximum removal was obtained after 8 h. Biomass particles of smaller size (90 microm) gave maximum adsorption (99.2%) at 100 mg/l concentration. The adsorption capacity increased with increase in temperature and agitation speed and the optimum were determined as 45 degrees C at 120 rpm. Freundlich and Langmuir isotherms were used to evaluate the data and the regression constants were derived. The adsorption rate constant values (Kad) were calculated for different initial concentration of Cr ions and the sorption was found to be higher at lower concentration (100 mg/l) of metal ion.     

Magnetic dye-affinity beads for human serum albumin purification

Cibacron Blue F3GA was covalently attached onto magnetic poly(vinyl alcohol) (mPVAL) beads (100-150 μm in diameter) for human serum albumin (HSA) adsorption from human plasma. Despite low nonspecific adsorption of HSA on mPVAL beads, Cibacron Blue F3GA attachment significantly increased the HSA adsorption. The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma. Desorption of HSA from mPVAL beads was achieved by medium containing 1.0 M KSCN at pH 8.0. To test the efficiency of albumin adsorption from human serum, before and after albumin adsorption was demonstrated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses. HSA molecules could be reversibly adsorbed and desorbed 10 times with the magnetic beads without noticeable loss in their HSA adsorption capacity.