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1.
mBio ; 9(6)2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30482835

RESUMO

As oral poliovirus vaccine (OPV) causes vaccine-associated paralytic poliomyelitis, the polio endgame strategy introduced by the Global Polio Eradication Initiative calls for a phased withdrawal of OPV and an introduction of inactivated poliovirus vaccine (IPV). The introduction of IPV creates challenges in maintaining the cold chain for vaccine storage and distribution. Recent advances in lyophilization have helped in finding a temperature-stable formulation for multiple vaccines; however, poliovirus vaccines have yet to capture a stable, safe formula for lyophilization. In addition, efficient in vitro methods for antigen measurement are needed for screening stable vaccine formulations. Here, we report size exclusion high-performance liquid chromatography (SE-HPLC) as a reliable means to identify the leading lyophilized formulation to generate thermostable Sabin inactivated poliovirus vaccine (sIPV). High-throughput screening and SE-HPLC determined the leading formulation, resulting in 95% D-antigen recovery and low residual moisture content of sIPV following lyophilization. Furthermore, the lyophilized sIPV remained stable after 4 weeks of incubation at ambient temperature and induced strong neutralizing antibodies and full protection of poliovirus receptor transgenic mice against the in vivo challenge of wild-type poliovirus. Overall, this report describes a novel means for the high-throughput evaluation of sIPV antigenicity and a thermostable lyophilized sIPV with in vivo vaccine potency.IMPORTANCE Poliomyelitis is a highly contagious disease caused by the poliovirus. While the live attenuated OPV has been the vaccine of choice, a major concern is its ability to revert to a form that can cause paralysis, so-called vaccine-associated paralytic poliomyelitis. Therefore, the new endgame strategy of the Global Polio Eradication Initiative includes the introduction of an IPV. However, the feasibility of the use of current IPV formulations in developing countries is limited, because IPV is insufficiently stable to be purified, transported, and stored under unrefrigerated conditions. We successfully designed the sIPV for use in the dry state that maintains the full vaccine potency in animal models after incubation at ambient temperature. This report provides, for the first time, candidate formulations of sIPV that are stable at elevated temperatures.


Assuntos
Liofilização , Poliomielite/prevenção & controle , Vacina Antipólio de Vírus Inativado/isolamento & purificação , Vacina Antipólio de Vírus Inativado/efeitos da radiação , Tecnologia Farmacêutica , Temperatura , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Estabilidade de Medicamentos , Camundongos Transgênicos , Vacina Antipólio de Vírus Inativado/administração & dosagem , Vacina Antipólio de Vírus Inativado/imunologia
2.
Protein Sci ; 15(2): 304-13, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16385003

RESUMO

High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of approximately 42%-58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (approximately 70%-100%) to achieve refolding yields of approximately 55%-78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.


Assuntos
Arginina/farmacologia , Ditiotreitol/metabolismo , Dissulfeto de Glutationa/metabolismo , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Naftalenossulfonato de Anilina , Animais , Proteínas de Transporte , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Fosfatases de Especificidade Dupla , Humanos , Pressão Hidrostática , Fosfoproteínas Fosfatases , Conformação Proteica , Renaturação Proteica , Proteínas Tirosina Fosfatases , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Solubilidade
3.
J Pharm Sci ; 93(12): 3076-89, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15514986

RESUMO

Benzyl alcohol, an antimicrobial preservative, accelerates aggregation and precipitation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in aqueous solution. The loss of native monomer during incubation at 37 degrees C was determined by analysis of sample aliquots with size exclusion high performance liquid chromatography (SE-HPLC). Benzyl alcohol caused minor perturbation of the tertiary structure of the protein without changing its secondary structure, documenting that the preservative caused a minor shift in the protein molecular population toward partially unfolded species. Consistent with this conclusion, in the presence of benzyl alcohol the rate of H-D exchange was accelerated and the fluorescence of 1-anilinonaphthalene-8-sulfonic acid in the presence of rhIL1ra was increased. Benzyl alcohol did not alter the free energy of unfolding based on unfolding experiments in urea or guanidine HCl. With differential scanning calorimetry it was determined that benzyl alcohol reduced the apparent Tm of rhIL-1ra, but this effect occurred because the preservative lowered the temperature at which the protein aggregated during heating. Isothermal calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra is relatively weak and hydrophobically driven. Thus, benzyl alcohol accelerates protein aggregation by binding to the protein and favoring an increase in the level of partially unfolded, aggregation-competent species. Sucrose partially inhibited benzyl alcohol-induced aggregation and tertiary structural change. Sucrose is preferentially excluded from the surface of the protein, favoring most compact native state species over expanded aggregation-prone forms.


Assuntos
Álcool Benzílico/farmacologia , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/metabolismo , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/análise , Sialoglicoproteínas/análise , Soluções , Água/metabolismo
4.
Yakugaku Zasshi ; 123(11): 957-61, 2003 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-14631757

RESUMO

A successful development of therapeutic proteins requires a formulation optimal for long-term storage of the proteins. During storage and shipment, proteins are subjected to multiple stresses. Here we show that ciliary neurotrophic factor (CNTF) readily aggregates upon exposure to mechanical stress such as agitation and elevated temperature at 37 degrees C. Sucrose and lysine or arginine protect CNTF from heat stress, while detergents such as Tween20 and organic solvents such as propylene glycol (PG) are effective against agitation. Combination of the amino acids and PG protected the protein from both stresses. The results suggest the importance of combining additives, against multiple stresses, which may have negative as well as positive influence individually against one particular stress.


Assuntos
Adjuvantes Farmacêuticos , Fator Neurotrófico Ciliar , Preparações Farmacêuticas , Desnaturação Proteica , Dobramento de Proteína , Proteínas , Estresse Mecânico , Química Farmacêutica , Detergentes , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Temperatura Alta , Lisina , Polissorbatos , Propilenoglicol , Sacarose , Temperatura
5.
Protein Sci ; 12(6): 1252-61, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12761396

RESUMO

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native-state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native-state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far- and near-UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native-state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl-induced unfolding, increased in proportion to sucrose concentration. Native-state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange-resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large-scale than on small-scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native-state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.


Assuntos
Conformação Proteica/efeitos dos fármacos , Proteínas/química , Sacarose/farmacologia , Animais , Bovinos , Dicroísmo Circular , Citocromos c/química , Deutério/química , Cavalos , Hidrogênio/química , Dobramento de Proteína , Ribonuclease Pancreático/química , Ribonucleases/química , Espectrofotometria Infravermelho , Termodinâmica
6.
Protein Sci ; 12(5): 903-13, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12717013

RESUMO

We studied the non-native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (DeltaG(unf)) and osmotic second virial coefficient (B(22)), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large DeltaG(unf) and negative B(22)), the first step is rate-limiting, and increasing DeltaG(unf) (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B(22) values) the second step is rate-limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate-limiting.


Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Coloides , Dimerização , Estabilidade de Medicamentos , Humanos , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica
7.
Biotechnol Annu Rev ; 8: 55-84, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12436915

RESUMO

One of the challenges for the successful commercialization of therapeutic proteins is to maintain the safety and efficacy of the protein during the manufacturing process, storage, and administration. To achieve this, the purified form of the protein drug is usually "formulated" with carefully selected excipients. The operations that occur subsequent to protein purification, such as freezing of the purified protein bulk, thawing of the bulk, formulation (excipient addition), sterile filtration, filling, freeze-drying, and inspection are commonly referred as "formulation and fill-finish operations". This review is focused on the protein formulation and fill-finish operations, critical process parameters at each operation, and the process considerations required for maintaining safety and efficacy of the drug during manufacturing and storage. Since proteins have complex molecular structures that can influence the protein stability, the reader is first introduced to salient concepts related to protein structure. This is followed by a review of the possible protein-degradation mechanisms and how a variety of external factors can contribute to protein degradation during the in vitro processing of the protein drug. The reader is then introduced to each of the formulation and fill-finish operations mentioned above, the possible degradations during each unit-operation, and process considerations necessary to avoid those degradations.


Assuntos
Excipientes/química , Liofilização/métodos , Preparações Farmacêuticas/química , Proteínas/química , Tecnologia Farmacêutica/métodos , Química Farmacêutica/métodos , Qualidade de Produtos para o Consumidor , Dessecação , Desenho de Fármacos , Estabilidade de Medicamentos , Armazenamento de Medicamentos/métodos , Liofilização/normas , Oxirredução , Pós , Conformação Proteica , Desnaturação Proteica , Proteínas/normas , Controle de Qualidade , Proteínas Recombinantes/química , Propriedades de Superfície , Tecnologia Farmacêutica/normas , Temperatura
8.
Protein Sci ; 11(7): 1825-33, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12070334

RESUMO

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.


Assuntos
Imunoglobulina G/metabolismo , Polietilenoglicóis/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Etanercepte , Concentração de Íons de Hidrogênio , Medições Luminescentes , Iodeto de Potássio/metabolismo
11.
Biochemistry ; 41(20): 6422-31, 2002 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-12009905

RESUMO

We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.


Assuntos
Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos/química , Precipitação Química , Dicroísmo Circular , Deutério/química , Dimerização , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Humanos , Hidrogênio/química , Cinética , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes , Soluções , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Sacarose/química , Propriedades de Superfície , Termodinâmica
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