Development and application of thermo-sensitive immunomicrospheres for antibody purification

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum. (c) 1994 John Wiley & Sons, Inc.     

BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states

The secondary structure and the thermostability of bovine serum albumin (BSA), before adsorption and after homomolecular displacement from silica and polystyrene particles, are studied by circular dichroism spectroscopy and differential scanning calorimetry. The structural perturbations induced by the hydrophilic silica surface are reversible, i.e. BSA completely regains the native structure and stability after being exchanged. On the other hand, the adsorption on, and subsequent desorption from, polystyrene particles causes irreversible changes in the stability and (secondary) structure of BSA. The exchanged proteins have a higher denaturation temperature and a lower enthalpy of denaturation than native BSA. The alpha-helix content is reduced while the beta-turn fraction is increased in the exchanged molecules. Both effects are more pronounced when the protein is displaced from less crowded sorbent surfaces. The irreversible surface-induced conformational change may be related to some aggregation of BSA molecules after being exposed to a hydrophobic surface.     

Development and application of thermo-sensitive magnetic immunomicrospheres for antibody purification

Ultrafine magnetite particles were prepared by a co-precipitation method. The poly-(styrene/N-isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. The minimum NaCl concentration for flocculation of these magnetic latex particles (critical flocculation concentration, CFC) decreased with increasing temperature. These temperature dependence of CFC, namely its thermo-sensitivity, originated from NIPAM. At a certain NaCl concentration, some of the magnetic latex particles showed reversible transition between flocculation and dispersion by controlling the temperature, and the thermo-flocculated magnetic latex particles were separated quickly in a magnetic field. Bovine serum albumin (BSA) was covalently immobilized onto the magnetic P(St/NIPAM/MAA) latex particles with high efficiency by the carbodiimide method. These thermo-sensitive magnetic immunomicrospheres were effective for the immunoaffinity purification of anti-BSA antibodies from antiserum.Correspondence to: A. Kondo     

Experimental collision efficiencies of polymer-flocculated animal cells.

The determination of particle collision kinetics is useful to decouple the effects of process parameters on individual events in flocculation. This paper discusses the effects of flocculation conditions on the collision efficiency of ATCC strain CRL 1606 hybridomas flocculated with poly-L-histidine. Experimental determinations of the collision efficiency of cells in Couette flow are presented over a range of experimental conditions. The collision efficiency correlates with the cell zeta potential to the -2.4 power at high surface coverage, consistent with literature results in latex systems. At low coverage, accounting for the distribution of polymer on the cells corrects for deviation from the high-coverage behavior. Collision is dependent on the hydrodynamic environment as well. At high surface coverage, collision efficiency is weakly dependent on hydrodynamic conditions and follows a dependency on the shear rate and viscosity to the -0.32 power. This is consistent with ionic coagulation theory. At low surface coverage, the collision efficiency is strongly dependent on the viscous fluid forces. The results versus both dose and shear rate over the entire range of surface coverages are consistent with weak intercell bonding. Collision kinetics in the presence of high molecular weight dextrans show steric hindrance to cell collision.     

Solute effects on the irreversible aggregation of serum albumin

Thermal stress on bovine serum albumin (BSA) promotes protein aggregation through the formation of intermolecular beta-sheets. We have used light scattering and chromatography to study effects of (<1 M) Na(2)SO(4), NaSCN, sucrose, sorbitol and urea on the rate of the thermal aggregation. Both salts were strong inhibitors of BSA aggregation and they reduced both the size and number (concentration) of aggregate particles compared to non-ionic solutes (or pure buffer). Hence, the salts appear to suppress both nucleation- and growth rate. The non-electrolyte additives reduced the initial aggregation rate (compared to pure buffer), but did not significantly limit the extent of aggregation in samples quenched after 27 min. heat exposure (40-50% aggregation in all samples). The non-electrolytes did, however, modify the aggregation process as they consistently brought about smaller but more concentrated aggregates than pure buffer. The results are discussed along the lines of linkage- and transition state theories. In this framework, the rate of the aggregation process is governed by the equilibrium between a thermally denatured state (D) and the transition state D( not equal). Thus, the effect of a solute relies on its preferential interactions with respectively D and D( not equal). The current results do not show any correlation between the solutes' preferential interactions with native BSA and their effect on the rate of aggregation. This suggests that non-specific, "Hofmeister-type" interactions, which scale with the solvent accessible surface area, are of minor importance. Rather, salt induced suppression of aggregation is suggested to depend on the modulation of specific electrostatic forces in the D( not equal) state.     

Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane

Generation of intestinal epithelial lipid polarity was studied in Caco-2 cells. Confluent monolayers on filters incorporated the exchangeable lipid N-6-NBD-aminocaproyl-sphingosine (C6-NBD-ceramide) from liposomes. The fluorescent ceramide was converted equally to C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, analogues of lipids enriched on the apical and basolateral surface, respectively, of intestinal cells in vivo. Below 16 degrees C, where vesicular traffic is essentially blocked, each fluorescent product accumulated in the Golgi area. At 37 degrees C, 50% had been transported to the cell surface within 0.5 h, as measured by selective extraction of the fluorescent lipids onto BSA in the medium ("back-exchange") at 10 degrees C. Transport to the two surfaces could be assayed separately, as a diffusion barrier existed for both NBD-lipids and BSA. C6-NBD-glucosylceramide was enriched twofold apically, whereas C6-NBD-sphingomyelin was equally distributed over both domains. Polarities did not decrease when 37 degrees C incubations were carried out in the presence of increasing BSA concentrations to trap the fluorescent lipids immediately after their arrival at the cell surface. Within 10 min from the start of synthesis, both products displayed their typical surface polarity. Lipid transcytosis displayed a half time of hours. In conclusion, newly synthesized sphingolipids in Caco-2 cells are sorted before reaching the cell surface. Transcytosis is not required for generating the in vivo lipid polarity.     

Thermal and metabolic responses to cold by men and by eumenorrheic and amenorrheic women

Previous work has suggested that men (M) are more sensitive to cold stress than women. There have also been observations that suggest that amenorrheic women (AW) are less thermally responsive than eumenorrheic women (EW). We investigated the hypothesis that M, EW, and AW would have different responses to cold stress. The subjects (6/group) were tested four times: twice at rest for 60 min (5 and 22 degrees C) and twice in a progressive exercise test (5 and 22 degrees C). At rest at 22 degrees C AW had a lower O2 uptake (VO2) than M and lower rectal (Tre) and finger temperatures than EW. At rest at 5 degrees C both AW and EW had lower skin temperature (Tsk) than M, but there were no group differences in peripheral Tsk sites. M increased VO2 after 10 min and EW after 20 min of cold stress; however, AW did not increase metabolism until 60 min. In the two exercise tests Tre increased in proportion to relative work load; in the 5 degrees C test there was little evidence that exercise increased Tsk sites above rest levels. Few of the metabolic or thermal differences could be accounted for by body fatness, body surface area (BSA), or BSA/kg. The data support the hypothesis that M, EW, and AW have different responses to cold stress.     

Effect of phase transition on the distribution of membrane-associated particles in microsomes.

(1) Rat liver microsomes were studied by freeze-fracture electron microscopy. The distribution of membrane-associated particles indicated the right-side-out orientation of microsomal vesicles. Studies at different temperatures were performed. At 30 degrees C membrane-associated particles are randomly distributed on membrane A-faces, while aggregations of particles are observed at 4 degrees C. (2) Aggregation is dependent on the cooling rates. It can be prevented by shock-freezing. (3) Particle aggregation is also prevented by cholesterol, added to the microsomes in equal molar ratio to the microsomal phospholid content. (4) These findings suggest that particle aggregation is caused by a partial freezing-out of phospholipid molecules during the phase transition from the liquid-crystalline to the gel state. (5) The results are discussed with respect to an observed increase in activation energy of microsomal drug monooxygenation at lower temperature.     

Flow cytometry: a promising technique for the study of silicone oil-induced particulate formation in protein formulations

Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also used to investigate the effects of silicone oil emulsions on the stability of BSA, lysozyme, abatacept, and trastuzumab formulations containing surfactant, sodium chloride, or sucrose. To aid in particle characterization, the fluorescence detection capabilities of flow cytometry were exploited by staining silicone oil with BODIPY 493/503 and model proteins with Alexa Fluor 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. The addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations.     

Adsorption of biopolymers at hydrophilic cellulose-water interface

The extent of adsorption (Gamma2(1)) of bovine serum albumin (BSA), beta-lactoglobulin, lysozyme, gelatin, and DNA from aqueous solution onto the hydrophilic surface of cellulose has been measured as function of biopolymer concentration at different temperatures, pHs, and ionic strengths, and in the presence of a high concentration of inorganic salts and denaturants. In all cases, the value of Gamma2(1) increases with the increase of biopolymer concentration (X2) in bulk and it attains a maximum value at a critical mole fraction concentration X2m. The value of Gamma2m depends upon the nature of protein, temperature, pH, and ionic strength, as well as the nature of neutral salts present in excess. Gamma2m for proteins at a fixed physicochemical condition stands in the following order: Gelatin>betalactoglobulin>lysozyme>BSA. The isotherms for adsorption of DNA nucleotides on cellulose surface at pH 4.0 have been compared at different temperatures and ionic strengths, and in the presence of high concentration of inorganic salts LiCl, NaCl, KCl, and Na2SO4. Values of Gamma2m for different systems have been evaluated and critically compared. At pH 6.0 and 8.0, Gamma2(1) values of DNA nucleotides on cellulose are all negative due to the excess positive hydration of cellulose. At pH 4.0, adsorption of nucleotides of acid, alkali, and heat-denatured DNA widely differ from each other and in the presence of excess concentration of urea becomes negative. The probable mechanisms of biopolymer-cellulose adsorption in terms of polymer hydration, steric interaction, London-van der Waals, hydrophobic, and other types of interactions have been discussed qualitatively. The standard free energy change for the adsorption of protein and DNA nucleotides on the cellulose surface at the state of adsorption saturation has been calculated in kJ per kg of cellulose using an integrated form of the Gibbs adsorption equation. The relation between DeltaG degrees and maximum affinities between biopolymers and the polysaccharide interface have been discussed for various systems.     

Protamine assembled in multilayers on colloidal particles can be exchanged and released

Biocomposite thin films assembled on colloidal particles by means of layer-by-layer adsorption have been suggested as drug carriers and diagnostic devices. Protamine (PRM)/dextransulfate (DXS) and protamine/bovine serum albumine (BSA) multilayers were fabricated on colloidal silica and subsequently investigated by means of fluorescence activated cell sorting (FACS) and microelectrophoresis. Fluorescein labeled polyelectrolytes were embedded at different positions in the multilayers as a marker for layer growth. FACS showed that PRM and DXS formed regular growing stable multilayers, yet adsorbed PRM can be nevertheless exchanged with PRM in solution during layer formation and also after the multilayer formation has been completed. Up to 90% of the PRM pool was available for exchange. PRM together with BSA as demonstrated by SFM did not form multilayers under the applied conditions although the zeta-potential, commonly used as an indicator for stepwise adsorption, observed characteristic alternations. The capability of bound PRM to exchange with PRM in solution is attributed to its relatively small size. The demonstrated exchange may have importance in designing multilayers with smart release features. Furthermore, FACS proved to be a rather suitable means to quantify the aggregation behavior during coating and washing. Singulets, doublets, triplets, and aggregates of higher order could be clearly resolved. The aggregation of particles coated with PRM/DXS layers was higher than that of silica particles coated with PAH/PSS layers. In the first case about 50% of all recorded events are attributed to aggregats, while the PAH/PSS coating produced only about 10% aggregates.     

Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.     

Formaldehyde-mediated aggregation of protein antigens: comparison of untreated and formalinized model antigens

A formaldehyde-mediated aggregation pathway (FMAP) is suggested as being primarily responsible for the aggregation of lyophilized tetanus toxoid (TT; a formalinized antigen) in the presence of moisture. The general occurrence of the FMAP was examined by using bovine serum albumin (BSA) and ribonuclease A (RNase) as model antigens; both protein antigens were formalinized according to a method commonly used to detoxify bacterial toxins. To clearly delineate the FMAP from other aggregation mechanisms, the aggregation kinetics and mechanism of both unmodified antigens (BSA and RNase) and formalinized antigens (f-BSA and f-RNase) were evaluated. We report that formaldehyde treatment introduces more rapid and extensive aggregation in antigens under conditions that favor the FMAP (i.e., 80% relative humidity and 37 degrees C). Consistent with formaldehyde-mediated crosslinking, f-antigen aggregates were covalent and non-disulfide-bonded, whereas BSA aggregates were disulfide-linked and RNase even did not aggregate under the same conditions. Coincorporation of amino acids (histidine and lysine), which strongly interact with formaldehyde, as well as prior antigen reduction with cyanoborohydride, significantly inhibited f-BSA aggregation, but showed no selective effect on BSA aggregation. Mechanistic analysis of f-BSA aggregates, inhibition studies, and similar reactivity of f-BSA with TT all confirmed the existence of the FMAP at moisture levels intermediate between the dry and solution state. This study demonstrates the potential for covalent reactions between formalinized protein antigens and neighboring chemical or biochemical species even after formalinization, and provides a general approach to inhibit the FMAP.     

Some factors determining protein aggregation during ultrafiltration

The factors contributing to protein aggregation in albumin ultrafiltration were investigated as a function of operation conditions. The nature of protein deposits was examined by electron microscopy. Protein aggregation appears to occur as a result of rapid supersaturation of protein molecules and high solvent velocity (shear) in the concentrated layer near the membrane surface. The shear occurring in the solvent flow on the membrane surface probably unfolds protein molecules and thus promotes flocculation due to collision between particles. (c) 1993 John Wiley & Sons, Inc.     

SEPARATION OF TWO FACTORS AFFECTING THE AGGREGATION KINETICS FROM THE CONDITIONED MEDIUM

Two effective factors contained in medium "conditioned" with a large number of cultured cells were separated through Sephadex G-25 gel, by assaying the plating efficiency and growth rate of chicken embryonic cells in secondary culture. The first factor, which is contained in the heavy molecular weight fraction α, can promote plating efficiency of the inoculated cells at low density. The second is contained in the light molecular weight fraction β, and promotes the growth rate of sparsely cultured cells. The effects of these factors upon the aggregation kinetics of singly isolated cells were studied. The factor in the fraction α promotes aggregation to result in fast flocculation. The factor in the fraction β, while not effective by itself, affects the aggregation of the cells treated with the factor in α. In the co-presence of both α and β, a "lag" was found after the initial fast flocculation. The possible mechanisms of the interaction of these two factors on the cell surface are discussed.     

Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aggregation of tryptophanyl-tRNA synthetase depending on temperature. Study by a low-angle scatter x-ray method

By means of small angle X-ray scattering, an aggregation of beef pancreas Trp-tRNA synthetase (EC 6.1.1.2) was observed at physiological temperatures. A Trp-tRNA synthetase preparation which is homogeneous after PAGE in beta-ME-SDS was found to be heterogeneous in particle sizes even at low (4-8 degrees C) temperature. At heating up to 30-45 degrees C, the oligomer sizes increased as well as its proportion depending on the incubation time and temperature; very large aggregates were observed 10 times exceeding the sizes of initial particles. Cooling to 20 degrees C caused no disaggregation due to disulphide bond formation between associated subunits of Trp-tRNA synthetase. A hypothesis is proposed that the aggregation of bovine Trp-tRNA synthetase evaluated in vitro and not observed earlier with any aminoacyl-tRNA synthetases of unicellular organisms might serve as one of the mechanisms of its compartmentation in pancreas.     

Thermal aggregation of bovine serum albumin at different pH: comparison with human serum albumin

We report here a study on thermal aggregation of BSA at two different pH values selected to be close to the isoelectric point (pI) of this protein. Our aim is to better understand the several steps and mechanisms accompanying the aggregation process. For this purpose we have performed kinetics of integrated intensity emission of intrinsic and extrinsic dyes, tryptophans and ANS respectively, kinetics of Rayleigh scattering and of turbidity. The results confirm the important role played by conformational changes in the tertiary structure, especially in the exposure of internal hydrophobic regions that promote intermolecular interactions. We also confirm that the absence of electrostatic repulsion favours the disordered non-specific interactions between molecules and consequently affects the aggregation rate. Finally, the comparison between BSA and another relative protein, HSA, allows us to clarify the role of different domains involved in the aggregation process. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

The budding mechanism of spikeless vesicular stomatitis virus particles.

Virus particles, lacking the spike G-glycoproteins, are produced during infection of Vero cells with the vesicular stomatitis virus mutant ts045 at the restrictive temperature 39.5 degrees C. At this temperature the mutated G proteins are blocked in their intracellular transport in the endoplasmic reticulum. We have studied the role of the G proteins in the formation of these spikeless virus particles. The results showed that the spikeless particles contain a full complement of membrane anchors, derived from the carboxy-terminal end of the G protein. Our observations suggest that virus particles are formed at the restrictive temperature with G protein which is later cleaved to produce spikeless particles. We suggest that this is due to a leak of G protein to the cell surface at 39.5 degrees C where budding then takes place, presumably driven by a G protein C-terminal tail--nucleocapsid interaction.     

Membrane transport of anandamide through resealed human red blood cell membranes

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments at 0 degrees C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 +/- 0.023, and the rate constant of unidirectional flux from inside to outside is 0.361 +/- 0.023 s(-1). The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]o) increases with the square root of [BSA]o in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane very rapidly, within seconds. At a molar ratio of anandamide to BSA of <1, membrane binding of anandamide increases with increasing temperatures between 0 degrees C and 37 degrees C, and the equilibrium dissociation constants are in the nanomolar range. The nature of membrane binding and the mechanism of membrane translocation are discussed.