Razvoj i usporedba alternativnih metoda pročišćavanja adalimumaba izravno iz suspenzije stanične kulture

Research background. Protein A affinity chromatography is a well-established method currently used in the pharmaceutical industry. However, the high costs usually associated with chromatographic separation of protein A and the difficulties in continuous operation make the investigation of alternative purification methods very important. Experimental approach. In this study, extraction/back-extraction and precipitation/dissolution methods were developed and optimised. They were compared with protein A and cation exchange chromatography separations in terms of yield of monoclonal antibody (mAb) and amount of residual impurities, such as DNA and host cell proteins, and amount of mAb aggregates. For a comprehensive comparison of the different methods, experiments were carried out with the same cell-free fermentation broth containing adalimumab. Results and conclusions. Protein A and cation exchange chromatographic separations resulted in high yield and purity of adalimumab. The precipitation-based process resulted in high yield but with lower purity. The extraction-based purification resulted in low yield and purity. Thus, the precipitation-based method proved to be more promising than the extraction-based method for direct purification of adalimumab from harvested cell culture fluid. Novelty and scientific contribution. Although alternative purification methods may offer the advantages of simplicity and low-cost operation, further significant improvements are required to compete with the performance of chromatographic separations of adalimumab from true fermentation broth.Pozadina istraživanja. Afinitetna kromatografija pri kojoj se monoklonsko protutijelo veže za protein A je već dobro poznata metoda u farmaceutskoj industriji. Međutim, zbog visokih troškova izdvajanja proteina A pomoću kromatografije te poteškoća u provođenju kontinuiranog procesa važno je ispitati alternativne metode pročišćavanja. Eksperimentalni pristup. U ovome su radu razvijene i optimirane metode ekstrakcije sa ponovnom ekstrakcijom te taloženja i otapanja. Rezultati tih dvaju metoda, odnosno prinos monoklonskih protutijela, količina preostalih nečistoća, kao što su DNA i proteini stanica domaćina, te količina agregata monoklonskih protutijela, uspoređeni su s onima dobivenim afinitetnom kromatografijom na ionskom izmjenjivaču. Za sveobuhvatnu usporedbu različitih metoda, ispitivanja su provedena na identičnim hranjivim podlogama koje su sadržavale adalimumab. Rezultati i zaključci. Afinitetnom kromatografijom na ionskom izmjenjivaču dobiveni su veliki prinos i velika čistoća adalimumaba. Taloženjem su dobiveni veliki prinos, ali manja čistoća uzorka. Pročišćavanje pomoću ekstrakcije rezultiralo je manjim prinosom i manjom čistoćom uzorka. Zaključeno je da je metoda taloženja bolja od ekstrakcije za izravno pročišćavanje adalimumaba iz suspenzije stanične kulture. Novina i znanstveni doprinos. Iako alternativne metode imaju neke prednosti, kao što su jednostavna i jeftina primjena, neophodno ih je unaprijediti da bi dobiveni rezultati bili usporedivi s rezultatima dobivenim pročišćavanjem adalimumaba ionskom kromatografijom u hranjivoj podlozi

The Effect of Surfactants and pH Modifying Agents on the Dissolution and Permeation of Pimobendan

Solubility and permeability are key parameters for establishing in vitro-in vivo correlation for poorly water-soluble active pharmaceutical ingredients (APIs). Recent studies demonstrate that not only solubility, but also effective permeability of the API may change due to the addition of solubilizing agents, and there is a certain mathematical relation between these physicochemical parameters. The aim of this study was to show the importance of early screening of solubility and permeability in presence of additives in order to achieve the expected bioavailability of the API. In this work, the effect of surfactants and microenvironmental pH modifiers were in focus, and pimobendan was chosen as model drug.In the case of pH modifiers, the equilibrium solubility of the API increased, while the permeability decreased significantly. No negative effect was observed for two surfactants at low additive levels, but these two additives also exhibited a slightly negative effect on permeability when used at higher concentrations. In the simultaneous dissolution-permeation studies the surfactants-containing formulation was found to have slightly higher flux than the pH-modifier-containing one. It can be due to the phenomenon that the dissolution of the active substance can be enhanced by these surfactants without any significant permeability reducing effect.The results obtained from the present study clearly demonstrate the importance of studying drug-additive interactions in every step of formulation development and based on these, the selection of the appropriate quality and quantity of additives. In addition, the results also underline the significance of performing simultaneous dissolution-permeation studies to predict bioavailability

Melt‐Blown and Electrospun Drug‐Loaded Polymer Fiber Mats for Dissolution Enhancement: A Comparative Study

Melt blowing (MB) was investigated to prepare a fast dissolving fibrous drug-loaded solid dispersion and compared with solvent-based electrospinning (SES) and melt electrospinning (MES). As a conventional solvent-free technique coupled with melt extrusion and using a high-speed gas stream, MB can provide high-quality micro- and nanofibers at industrial throughput levels. Carvedilol, a weak-base model drug with poor water solubility, was processed using a common composition optimized for the fiber spinning and blowing methods based on a hydrophilic vinylpyrrolidone-vinyl acetate copolymer (PVPVA64) and PEG 3000 plasticizer. Scanning electron microscopy combined with fiber diameter analysis showed diameter distributions characteristic to each prepared fibrous fabrics (the mean value increased toward SES<MB<MES). Differential scanning calorimetry and X-ray diffraction studies revealed that the incorporated drug was in amorphous form regardless the preparation method. The HPLC studies demonstrated that all of the materials produced by the different techniques passed the regulatory purity requirements. The fibers exhibited ultrafast drug release tested under neutral pH conditions; the melt-blown sample dissolved within 2 min owing to its large specific surface area. The presented results confirm the applicability of MB as a novel formulation technique for polymer-based drug delivery systems