Biomedical subjects

Y F Maa

Publications and source records attributed to Y F Maa.

12 recordsLinked to original sources

PubMed1998

Spray-drying of air-liquid interface sensitive recombinant human growth hormone.

Spray-drying is an attractive method for preparing fine recombinant human growth hormone (rhGH) powders if the detrimental effect of protein degradation at the air-liquid interface on the protein can be minimized. In this study, we demonstrated that rhGH degradation (insoluble and soluble aggregate formation), as the consequence of air-liquid interfacial degradation, could be prevented using the appropriate formulation. Adding polysorbate-20 surfactant into the liquid feed (with no presence of sugar protectant) significantly reduced the formation of insoluble protein aggregates, while adding the divalent metal zinc ion effectively suppressed the formation of soluble protein aggregates. The combination of the two yielded a spray-dried rhGH powder having insignificant protein degradation. Our data suggest that the two components might protect the protein through different mechanisms. Polysorbate molecules occupy the air-liquid interface of spray droplets, thereby reducing the chance for rhGH to form insoluble aggregates by surface denaturation. Two zinc ions associate with two rhGH molecules to form a dimer complex that can resist the formation of soluble protein aggregates. Characterization of spray-dried powders by scanning electron microscopy suggests that both formulation and drying conditions have a strong influence on particle morphology and shape. Overall, spherical rhGH powders of smooth surface and good biochemical quality can be prepared by spray-drying using this formulation with no addition of sugar protectant.

Calorimetry, Differential Scanning↗

PubMed1998

Effect of mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody.

We have examined the stability and aerosol performance of the pharmaceutical protein recombinant humanized anti-IgE monoclonal antibody (rhuMAbE25) spray dried with mannitol. The aerosol performance was measured by the fine particle fraction (FPF), and stability was assessed by the formation of soluble aggregates. When mannitol was added to the spray-dried rhuMAbE25 formulation, its ability to stabilize the protein leveled off above about 20% (w/w, dry basis). The FPF of the spray-dried formulations was stable during storage for rhuMAbE25 containing 10% and 20% mannitol, but the 30% formulation exhibited a dramatic decrease upon storage at both 5 degreesC and 30 degreesC, due to mannitol crystallization. We tested the addition of sodium phosphate to a 60:40 rhuMAbE25:mannitol (w:w) mixture, which otherwise crystallized upon spray drying and yielded a nonrespirable powder. The presence of sodium phosphate was successful in inhibiting mannitol crystallization upon spray drying and dramatically lowering the rate of solid-state aggregation. However, over long-term storage some crystallization was observed even for the phosphate-containing samples, concomitantly with increased particle size and decreased suitability for aerosol delivery. Therefore, the physical state of mannitol (i.e., amorphous or crystalline) plays a role both in maintaining protein stability and providing suitable aerosol performance when used as an excipient for spray-dried powders. Agents which retard mannitol crystallization, e.g., sodium phosphate, may be useful in extending the utility of mannitol as an excipient in spray-dried protein formulations.

Antibodies, Anti-Idiotypic↗

PubMed1998

Investigation on fouling mechanisms for recombinant human growth hormone sterile filtration.

During sterile filtration of recombinant human growth hormone solutions, severe membrane fouling was experienced compared to other protein preparations of significantly higher molecular weights and concentrations. This phenomenon was attributed to rhGH aggregation/adsorption occurring in the filter pore. To better understand this phenomenon, we examined several possible fouling mechanisms: (1) pore constriction, (2) adsorption due to nonspecific binding between protein and the membrane, (3) shear-induced adsorption, (4) hydrophobic interface-induced aggregation/adsorption. The protein solutions were sterily filtered using 0.22 mm filters, and their filtration fluxes were monitored. Filtration on the capillary and the noncapillary filters suggested that constraints by pore constriction and tortuosity played only a limited role. Filtration using filters with different degrees of protein binding tendency suggested that nonspecific adsorption was insignificant. The shear stress acting on the protein during filtration was small. RhGH which was intentionally sheared in a high-speed concentrically rotating device did not aggravate fouling tendency, suggesting that the shear-induced adsorption might not be the major fouling mechanism. The dynamic light scattering data showed a trace amount of rhGH aggregates always present in equilibrium with the hydrophobic (air-water and membrane-water) interface. These aggregates tended to be adsorbed to the membrane, and more aggregates were generated presumably due to the equilibrium between aggregates and protein monomers. This adsorption/aggregation process eventually fouled the membrane. When the hydrophobic interface was occupied by surfactant molecules, the equilibration kinetics ceased to generate aggregates, thereby minimizing membrane fouling. This study clarified the cause of such an unusual fouling phenomenon upon microfiltration.

Adsorption↗

PubMed1998

Effect of spray drying and subsequent processing conditions on residual moisture content and physical/biochemical stability of protein inhalation powders.

PURPOSE: To understand the effect of spray drying and powder processing environments on the residual moisture content and aerosol performance of inhalation protein powders. Also, the long-term effect of storage conditions on the powder's physical and biochemical stability was presented. METHODS: Excipient-free as well as mannitol-formulated powders of a humanized monoclonal antibody (anti-IgE) and recombinant human deoxyribonuclease (rhDNase) were prepared using a Buchi 190 model spray dryer. Residual moisture content and moisture uptake behavior of the powder were measured using thermal gravimetric analysis and gravimetric moisture sorption isotherm, respectively. Protein aggregation, the primary degradation product observed upon storage, was determined by size-exclusion HPLC. Aerosol performance of the dry powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger (MSLI). RESULTS: Spray-dried powders with a moisture level (approximately 3%) equivalent to the freeze-dried materials could only be achieved using high-temperature spray-drying conditions, which were not favorable to large-male manufacturing, or subsequent vacuum drying. These dry powders would equilibrate with the subsequent processing and storage environments regardless of the manufacturing condition. As long as the relative humidity of air during processing and storage was lower than 50%, powders maintained their aerosol performance (fine particle fraction). However, powders stored under drier conditions exhibited better long-term protein biochemical stability. CONCLUSIONS: Manufacturing, powder processing, and storage environments affected powder's residual moisture level in a reversible fashion. Therefore, the storage condition determined powder's overall stability, but residual moisture had a greater impact on protein chemical stability than on powder physical stability.

Administration, Inhalation↗

PubMed1997

The effect of operating and formulation variables on the morphology of spray-dried protein particles.

The purpose of this research was to investigate the shape and morphology of various spray-dried protein powders as a function of spray-drying conditions and protein formulations. A benchtop spray dryer was used to spray dry three model proteins in formulation with a sugar or a surfactant. Physical characterizations of the powder included morphology (scanning electron microscopy), particle size, residual moisture, and X-ray powder diffraction analyses. A significant change in particle shape from irregular (e.g., "donut") to spherical was observed as the outlet temperature of the dryer was decreased. The drying air outlet temperature was shown to depend on various operating parameters and was found to correlate with the drying rate of atomized droplets in the drying chamber. The morphology of spray-dried protein particles was also affected by formulation. In protein:sugar formulations, spray-dried particles exhibited a smooth surface regardless of the protein-to-lactose ratio, whereas roughness was observed when mannitol was present at > 30% of total solids, due to recrystallization. Protein particles containing trehalose at concentrations > 50% were highly agglomerated. The presence of surfactant resulted in noticeably smoother, more spherical particles. The shape and the morphology of spray-dried powders are affected by spray drying conditions and protein formulation. This study provides information useful for development of dry proteins for fine powder (e.g., aerosol) applications.

Antibodies, Monoclonal↗

PubMed1990

Rapid high-performance liquid chromatography of nucleic acids with polystyrene-based micropellicular anion exchangers.

Nucleic acids were separated by ion-exchange chromatography on 30 x 4.6 and 100 x 4.6 mm columns packed with a micropellicular anion exchanger made of 3-microns rigid polystyrene-based non-porous microspheres with a covalently bound hydrophilic layer and DEAE functional groups at the surface. The stationary phase particles showed negligible swelling in methanol according to permeability measurements with water and methanol. Nucleic acids and their fragments including synthetic single-stranded oligonucleotides, linear, nicked and supercoiled DNAs as well as DNA restriction fragments were separated in less than 5 min, a time scale that is much smaller than that of conventional high-performance liquid chromatographic analysis for such samples. When only buffer and sodium chloride were used in the eluent for the separation of double-stranded DNA restriction fragments pGEM-3Z/Taq I, electrophoretic analysis of the effluent revealed the presence of smaller fragments in the bands of the larger ones. Upon addition of ethylenediaminetetraacetic (EDTA) salt to the eluent, however, such contamination by shorter fragments was no longer observed. In the absence of EDTA, magnesium chloride in the eluent at a concentration of 1 mM precluded the separation of the restriction fragments under otherwise identical chromatographic conditions.

Anion Exchange Resins↗

PubMed1988

Mixed-bed ion-exchange columns for protein high-performance liquid chromatography.

Protein retention is investigated on high-performance liquid chromatography columns packed with mixtures of ion exchangers. Retention factors are measured at both low and high salt concentrations in the eluent and their dependence on the bed composition is found to be linear in some cases, but non-linear in others. The physical basis for the observed non-linear retention behavior has not been established and an empirical mixing rule is employed to express the dependence of protein retention on bed composition. Protein separations are carried out on the mixed-bed columns by using gradient elution with increasing salt concentration and the process is modelled mathematically. The retention times predicted by computer calculations correspond closely to the experimental findings. Optimal selection of the mixed-bed composition and the gradient steepness for the separation of four proteins is illustrated by using the window diagram technique. Although the experimental results presented here deal with electrostatic interaction chromatography of proteins only the applicability of mixed sorbents is expected to extend to all branches of liquid chromatography. It is anticipated that mixed-sorbent columns will find extensive use in the large-scale purification of biological compounds and in routine analysis.

Ammonium Sulfate↗

PubMed1988

Rapid analysis of proteins and peptides by reversed-phase chromatography with polymeric micropellicular sorbents.

Peptides and proteins were separated by reversed-phase chromatography on a 30 x 4.6 mm I.D. column packed with non-porous crosslinked polystyrene particles having a mean particle diameter of 3 micron and a rugulose surface. The polymeric support did swell slightly in organic solvents, but the estimated 5-8% change in particle diameter did not adversely affect the efficiency of the column which was used repeatedly with gradient elution from water to organic solvent under conditions typically employed in reversed-phase chromatography. In these experiments, the pH of the eluent was varied in a wide range in order to compare the effect of acidic and alkaline eluents on the separation of protein and complex peptide mixtures. The column showed no deterioration even after extensive exposure to alkaline mobile phases. The retention behavior of sixteen proteins having widely different pI values was studied as a function of the eluent pH. The chromatographic system exhibited large selectivity differences upon changing the pH of the eluent from 2 to 11. Analytical information about peptide and protein mixtures could therefore be enhanced by using eluents at the pH extremes. At the pH extremes of 2 and 11 peak sharpness and protein mass recovery were found to be superior to those obtained with neutral eluents. Usually the column temperature was held at 80 degrees C and typical analysis times ranged from 30 s to 10 min as illustrated by chromatograms of protein mixtures and by peptide maps. With regular use under such conditions the column showed no deterioration after three months.

Chromatography, High Pressure Liquid↗

PubMed1988

High-performance liquid chromatography with concanavalin A immobilized by metal interactions on the stationary phase.

Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.

Carbohydrates↗

PubMedDate not supplied

Microencapsulation reactor scale-up by dimensional analysis.

A microencapsulation process for preparing protein-loaded microspheres based on a solvent-extraction method was scaled up using continuously stirred tank reactors (CSTR) from 1 L to 100 L in batch size. This study was concerned with developing a quantitative correlation between the size of the microspheres and process parameters. The process parameters considered include operational variables and physical properties associated with both the dispersion and dispersed phases. Dimensional analysis was used to establish such a correlation based on protein-free poly(lactic acid-co-glycolic acid) microspheres in an oil-in-water emulsion system prepared in a 1 L CSTR. This correlation was found to accurately describe the preparation of protein-loaded microspheres in a solid-in-oil-in-water system. Poly(methyl methacrylate) was found to behave similarly to poly(lactic-acid-co-glycolic acid) and could be used as a model polymer for scale-up investigation. This study showed that dimensional analysis can be used to predictably scale the current microencapsulation process up to 100 L to produce particles of defined size.

Carboxymethylcellulose Sodium↗

PubMedDate not supplied

Liquid-liquid emulsification by static mixers for use in microencapsulation.

4e report a feasibility study on liquid-liquid emulsification by static mixers for use in microencapsulation. This study was concerned with developing a quantitative correlation between the size of the microspheres and process parameters. The process parameters considered included operational variables and physical properties associated with both the dispersion and dispersed phases. The effect of mixing element design on mixing efficiency and particle-size distribution was evaluated using three different static mixers. The performance of static mixers and conventional continuously stirred-tank reactors was assessed in several different aspects. Dimensional analysis was used to establish a correlation based on protein-free poly(lactic acid-co-glycolid acid) and poly(methyl methacrylate) microspheres in an oil-in-water emulsion using 1/4", 1/2" and 1" static mixers. This correlation accurately described the preparation of protein-loaded microspheres and provided large-scale microsphere production with a empirical basis.

Biocompatible Materials↗

PubMedDate not supplied

Effect of primary emulsions on microsphere size and protein-loading in the double emulsion process.

Incorporation of a protein drug in microspheres made of a hydrophobic polymer is commonly achieved via double liquid-liquid emulsification (w/o/w) or by dispersing a powdered protein in a polymer solution followed by liquid-liquid emulsification (s/o/w). This study focused on the effect of the first operating step in both processes on the size and protein-loading of the microspheres. Bovine serum albumin (BSA) was used as the model protein and poly(methyl methacrylate) (PMMA) was used as the model polymer. The w/o emulsion was characterized based on the degree of emulsion fineness which was controlled using rotor/stator homogenization. The s/o emulsion was characterized based on protein powder size and shape. Protein powders of different sizes and shapes were produced using different powder preparation methods. In both emulsification processes, the second operating step which produced the microspheres was conducted in either a continuously stirred tank reactor (CSTR) or a static mixer. The size of the microspheres thus prepared was found to increase with increasing size of the protein powder in the s/o/w system but increase with decreasing size of the liquid emulsion droplets in the w/o/w system. Empirical correlations can accurately predict the size of the microspheres if the size of w/o emulsion droplets and protein powder is 10 x less than the microsphere size. Protein loading in the microspheres decreased with respect to increases in w/o emulsion droplet size or in protein powder size. We propose that these phenomena are attributed to two mechanisms, fragmentation along the weak routes in the w/o/w system and particle redistribution as the result of terminal velocity in the s/o/w system. The role of protein powder shape was not significant until the protein powder size exceeded 5 microns. Irregular-shaped protein powders resulted in lower encapsulation efficiency than spherical-shaped protein powders.

Animals↗