RESUMEN
Host-cell lipases can be present in monoclonal antibody drug products and can degrade polysorbates present in the formulations as stabilizers. We hypothesized that the in-use stability of the IV admixture prepared from such a drug product might be impacted by decreasing levels of polysorbate 20. Host-cell lipase activity has, in fact, been observed during development of one of our therapeutic monoclonal antibody drug products. Throughout the course of the product shelf life, polysorbate 20 levels decreased but no other quality attributes of the drug product were impacted. An experimental approach was developed to simulate how the prepared IV admixture in-use stability is affected as polysorbate 20 concentration in the drug product decreased over the shelf life, and from that a minimum level of polysorbate 20 required in the drug product was determined to estimate the in-use stability of the IV admixture as the polysorbate 20 in the drug product degrades. The results indicate that although the observed degradation of polysorbate 20 does not affect quality attributes of this drug product, in-use stability of the IV admixture as a function of polysorbate degradation can be impacted and should be assessed to ensure sufficient quality.
Asunto(s)
Lipasa , Polisorbatos , Polisorbatos/metabolismo , Composición de Medicamentos , Anticuerpos Monoclonales/metabolismoRESUMEN
Polysorbates (PSs) are a class of surfactants commonly used in the formulation of protein therapeutic agents to provide protection against denaturation and aggregation. When the PS in these drug formulations degrades, loss of stabilization of the protein therapeutic and formulation may occur, resulting in particulate formation or other undesirable changes in product critical quality attributes. Here, we present a simplified platform to predict long-term PS20 and PS80 degradation for monoclonal antibody drugs containing the PS-degrading enzyme lysosomal acid lipase. The platform was based on a temperature-dependent equation derived from existing PS20 degradation stability data. Accurate prediction of both PS20 and PS80 hydrolysis for as long as 2 years was achieved through short-term kinetics studies performed within 2 weeks. This platform substantially shortens the time required to determine the long-term stability of PS degradation and therefore can be used to guide the purification process and optimization of antibody formulations.
Asunto(s)
Polisorbatos , Tensoactivos , Polisorbatos/metabolismo , Cinética , Hidrólisis , TemperaturaRESUMEN
PURPOSE: Polysorbates (PS) are excipients used in the biotech industry to stabilize monoclonal antibody (mAb) protein products. However, PS in drug product formulations can be degraded during storage and lead to particle formation because of the limited solubility of the free fatty acids released through the enzymatic hydrolysis of PS-a process driven by residual host cell proteins, especially lipases, that are co-purified with the drugs. When multiple lipases are present, it is very difficult to know the cause for PS degradation. In this study, we aim to determine the cause of PS degradation from two lipases, lysosomal acid lipase (LAL) and lipoprotein lipase (LPL). METHODS: PS degradation pattern of the drug product was compared with those induced by recombinant lipases. Correlations between the concentration of LPL or LAL and PS20 loss were compared. Specific inhibitors, LAL inhibitor lalistat2 and LPL inhibitor GSK264220A, were used to differentiate their degradation of PS in the drug products. RESULTS: The complete inhibition of PS20 degradation by lalistat2 suggested that LAL, rather than LPL, was responsible for the PS20 degradation. In addition, LAL was more strongly correlated than LPL with the percentage of PS20 degradation. No PS20 degradation was observed for several mAbs containing similar levels of LPL (0.5-1.5 ppm) in the absence of LAL, suggesting that LPL concentrations below 1.5 ppm does not degrade PS20 in drug products. CONCLUSIONS: LAL was determined to be the cause of the PS20 degradation. This study provides a practical strategy to determine the root cause of PS degradation.
Asunto(s)
Anticuerpos Monoclonales , Polisorbatos , Composición de Medicamentos , Solubilidad , TensoactivosRESUMEN
Polysorbates (PSs) are surfactants commonly added to therapeutic protein drug product formulations to protect proteins from denaturation and aggregation during storage, transportation, and delivery. However, enzymatic hydrolysis of PSs has been recognized as the primary route of PS degradation in monoclonal antibody formulations, resulting in the release of free fatty acids that drive undesired particulate formation. Here, we present a rapid lipase activity assay with optimized incubation conditions for accurate quantitation of free fatty acids without a fatty acid extraction step. This assay can detect low levels of PS degradation (0.000024% PS20 degradation) within 1 day with minimal sample preparation. The levels of released free fatty acids were found to strongly correlate with the degree of PS20 degradation. The case study described herein suggests that this approach can detect low levels of PS20 degradation caused by sub-ppm lipase levels within 1 day, compared with the duration of 14 days needed for PS degradation assays based on two-dimensional liquid chromatography-charge aerosol detection.
Asunto(s)
Anticuerpos Monoclonales/química , Ácidos Grasos no Esterificados/análisis , Lipasa/química , Polisorbatos/química , Cromatografía Líquida de Alta Presión/métodos , Ácidos Grasos/análisis , Ácidos Grasos no Esterificados/química , Hidrólisis , Solubilidad , Tensoactivos/químicaRESUMEN
A simple method for interlocking glucose oxidase and horseradish peroxidase in a network of cellulose fibers coated with bovine serum albumin (BSA)-exfoliated graphene (biographene) is reported here. The resulting paper reactor is inexpensive and stable. Biographene is expected to function as an electron shuttle, making the reaction between the enzyme and the substrate more efficient, and this hypothesis is examined here. The BSA used to separate the sheets of graphene provides extra carboxylic acid groups and primary amines to help interlock the enzymes and the graphene in between the fibers. The decrease in entropy associated with interlocking the enzymes on a solid support is likely responsible for the increase in enzymatic stability/activity observed. Each cellulose disk contained 5.2mg of enzyme per gram of paper and 93% of the enzyme is retained after washing for 0.5-2h. This simple methodology provides a low cost, effective approach for achieving high enzymatic activity and good loadings on a benign, versatile support.
Asunto(s)
Enzimas Inmovilizadas/química , Glucosa Oxidasa/química , Grafito/química , Ingeniería de Proteínas/métodos , Celulosa/química , Peroxidasa de Rábano Silvestre , Albúmina Sérica Bovina/químicaRESUMEN
A potentially universal approach is presented for enzyme attachment to cellulose that significantly enhances enzyme stability while retaining high activity, and involves no chemical functionalization of cellulose. Bovine serum albumin (BSA) was interlocked in cellulose to form a protein-friendly surface (named BSA-Paper), while also providing COOH and NH2 groups for subsequent attachment of enzymes. The desired enzyme is then mixed with additional BSA and interlocked on BSA-Paper. The second BSA layer dilutes and crosslinks the enzyme for improved stability. Laccase was tested as a model enzyme for interlocking on BSA-Paper, and was found to retain over 100 % activity and was 240 times more stable at 25 °C (half life=180â d) than laccase. This new approach was also tested with a few other enzymes with encouraging results, thus providing a potentially universal method for stabilization of enzymes on cellulose with retention of high activities.
Asunto(s)
Celulosa/metabolismo , Lacasa/metabolismo , Papel , Albúmina Sérica Bovina/metabolismo , Animales , Bovinos , Celulosa/química , Colorimetría , Tamaño de la Partícula , Albúmina Sérica Bovina/química , Propiedades de SuperficieRESUMEN
A simple method for interlocking glucose oxidase (GOx) and horseradish peroxidase (HRP) in cellulose fibers using poly(acrylic acid) (PAA) as an armor around the enzyme, without any need for activation of the cellulose support, is reported here. The resulting enzyme paper is an inexpensive, stable, simple, wearable, and washable biosensor. PAA functions as a multifunctional tether to interlock the enzyme molecules around the paper fibers so that the enzymes are protected against thermal/chemical denaturation and not released from the paper when washed with a detergent. The decreased conformational entropy of the interlocked enzyme protected by the nanoarmor is likely responsible for increased enzyme stability to heat and chemical denaturants (retained ≥70 percent enzyme activity after washing with urea or SDS for 30min), and the polymer protects the enzyme against inactivation by proteases, bacteria, inhibitors, etc. The kinetics of the interlocked enzyme were similar to that of the enzyme in solution. The Vmax was 6(±0.5)mM per minute before washing, then increased slightly to 9(±1.4)mM per minute after washing with water. The Km was 22(±6.4mM), which was slightly higher compared to GOx in solution (25-27mM). Because the surface area of the paper does not limit the enzyme loading, about 20% of enzyme was successfully loaded onto the paper (0.2g enzyme per gram of paper), and ≥95% of the enzyme was retained after washing. Interlocking works with other enzymes such as laccase, where ≥60% of the enzyme activity is retained. This novel methodology provides a low cost, simple, modular approach of achieving high enzyme loadings in ordinary filter paper, not limited by cellulose surface area, and there has been no need for complex methods of enzyme engineering or toxic methods of activation of the solid support to prepare highly active biocatalysts.
Asunto(s)
Resinas Acrílicas/química , Celulosa/química , Enzimas Inmovilizadas/química , Técnicas Biosensibles , Reactivos de Enlaces Cruzados/química , Estabilidad de Enzimas , Glucosa Oxidasa/química , Peroxidasa de Rábano Silvestre/química , Papel , PorosidadRESUMEN
Several key properties of catalase such as thermal stability, resistance to protease degradation, and resistance to ascorbate inhibition were improved, while retaining its structure and activity, by conjugation to poly(acrylic acid) (PAA, Mw 8000) via carbodiimide chemistry where the amine groups on the protein are appended to the carboxyl groups of the polymer. Catalase conjugation was examined at three different pH values (pH 5.0, 6.0, and 7.0) and at three distinct mole ratios (1:100, 1:500, and 1:1000) of catalase to PAA at each reaction pH. The corresponding products are labeled as Cat-PAA(x)-y, where x is the protein to polymer mole ratio and y is the pH used for the synthesis. The coupling reaction consumed about 60-70% of the primary amines on the catalase; all samples were completely water-soluble and formed nanogels, as evidenced by gel electrophoresis and electron microscopy. The UV circular dichroism (CD) spectra indicated substantial retention of protein secondary structure for all samples, which increased to 100% with increasing pH of the synthesis and polymer mole fraction. Soret CD bands of all samples indicated loss of â¼50% of band intensities, independent of the reaction pH. Catalytic activities of the conjugates increased with increasing synthesis pH, where 55-80% and 90-100% activity was retained for all samples synthesized at pH 5.0 and pH 7.0, respectively, and the Km or Vmax values of Cat-PAA(100)-7 did not differ significantly from those of the free enzyme. All conjugates synthesized at pH 7.0 were thermally stable even when heated to â¼85-90 °C, while native catalase denatured between 55 and 65 °C. All conjugates retained 40-90% of their original activities even after storing for 10 weeks at 8 °C, while unmodified catalase lost all of its activity within 2 weeks, under similar storage conditions. Interestingly, PAA surrounding catalase limited access to the enzyme from large molecules like proteases and significantly increased resistance to trypsin digestion compared to unmodified catalase. Similarly, negatively charged PAA surrounding the catalase in these conjugates protected the enzyme against inhibition by negatively charged inhibitors such as ascorbate. While Cat-PAA(100)-7 did not show any inhibition by ascorbate in the presence of 270 µM ascorbate, unmodified catalase lost â¼70% of its activity under similar conditions. This simple, facile, and rational methodology produced thermostable, storable catalase that is also protected from protease digestion and ascorbate inhibition and most likely prevented the dissociation of the multimer. Using synthetic polymers to protect and improve enzyme properties could be an attractive approach for making "Stable-on-the-Table" enzymes, as a viable alternative to protein engineering.
Asunto(s)
Resinas Acrílicas/química , Catalasa/química , Animales , Catalasa/antagonistas & inhibidores , Catalasa/metabolismo , Bovinos , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , TemperaturaRESUMEN
The thermosolvatochromism of nitrospiropyran free and bound to cyclodextrin was studied in dimethylsulfoxide (DMSO)-water binary solvent systems. Spiropyran was interconverted to merocyanine by heating the sample to 55 °C. The merocyanine (MC) was converted back to spiropyran (SP) either by cooling the sample to room temperature or irradiating the sample with a visible light emitting diode. Steady state absorption spectra of SP and MC samples in the free state and bound to cyclodextrin were obtained in several DMSO-water binary solutions. Emission spectra of MC both free and cyclodextrin-bound were also acquired. Blue-shifted absorption and emission spectra of the studied molecules with increasing solvent polarity suggest that the dipole moments of free and bound merocyanines are higher in the ground state compared to the excited state. Merocyanine dipole moments in the ground and excited states were determined using thermosolvatochromism measurements and the Lippert-Mataga, Bakhshiev, and Kawski-Chamma-Viallet polarity functions. A large change in the dipole moment (ca. 16 D) of the merocyanine in aqueous DMSO was obtained upon electronic excitation, S(1) â S(0). Analysis of the merocyanine Stokes' shifts as a function of solvent polarity indicates that both general solvent effects and specific solvent effects are present in all systems studied. These findings reveal that merocyanine could be utilized as a polarity sensor for DMSO-water binary solutions.