Risperidone-Loaded PLGA-Lipid Particles with Improved Release Kinetics: Manufacturing and Detailed Characterization by Electron Microscopy and Nano-CT.

For parenteral controlled drug release, the desired zero order release profile with no lag time is often difficult to achieve. To overcome the undesired lag time of the current commercial risperidone controlled release formulation, we developed PLGA-lipid microcapsules (MCs) and PLGA-lipid microgels (MGs). The lipid phase was composed of middle chain triglycerides (MCT) or isopropylmyristate (IPM). Hydroxystearic acid was used as an oleogelator. The three-dimensional inner structure of Risperidone-loaded MCs and MGs was assessed by using the invasive method of electron microscopy with focused ion beam cutting (FIB-SEM) and the noninvasive method of high-resolution nanoscale X-ray computed tomography (nano-CT). FIB-SEM and nano-CT measurements revealed the presence of highly dispersed spherical structures around two micrometres in size. Drug release kinetics did strongly depend on the used lipid phase and the presence or absence of hydroxystearic acid. We achieved a nearly zero order release without a lag time over 60 days with the MC-MCT formulation. In conclusion, the developed lipid-PLGA microparticles are attractive alternatives to pure PLGA-based particles. The advantages include improved release profiles, which can be easily tuned by the lipid composition.

Origin of Aggregate Formation in Antibody Crystal Suspensions Containing PEG.

The crystalline state of proteins is deemed as a promising formulation platform for biopharmaceuticals. However, a stabilizing effect of protein crystal suspensions is controversially discussed. In fact, antibodies can display an increased aggregation and particle formation profile after the crystallization process compared with liquid or solid amorphous formulations. Nevertheless, studies regarding aggregate formation and its origin remain meager in literature. It was the aim of this study to investigate these aspects for a model IgG antibody (mAb1), which shows an increased aggregate formation after crystallization with polyethylene glycol. The presence of a dynamic equilibrium, a steady exchange of protein between the crystals and the supernatant, was demonstrated by replacing the supernatant with an identical but fluorescence-labeled protein solution and followed by confocal laser scanning microscopy. Aggregate formation was monitored by size exclusion high-pressure chromatography and flow cytometry. Constantly increasing aggregate levels were found for the crystal fraction and for the supernatant. For the later, markedly higher particle counts were detected. The labeled supernatant and the unlabeled protein crystals allowed a precise identification of the origin of the aggregates. The rising aggregate fractions of the crystals displayed high mean fluorescence intensities that elucidated their origin in the supernatant.

The "New Polyethylene Glycol Dilemma": Polyethylene Glycol Impurities and Their Paradox Role in mAb Crystallization.

Polyethylene glycols (PEG) represent the most successful and frequently applied class of excipients used for protein crystallization. PEG auto-oxidation and formation of impurities such as peroxides and formaldehydes that foster protein drug degradation is known. However, their effect on mAb crystallization has not been studied in detail before. During the present study, a model IgG1 antibody (mAb1) was crystallized in PEG solutions. Aggregate formation was observed during crystallization and storage that was ascribed to PEG degradation products. Reduction of peroxide and formaldehyde levels prior to crystallization by vacuum and freeze-drying was investigated for its effect on protein degradation. Vacuum drying was superior in removal of peroxides but inferior in reducing formaldehyde residues. Consequently, double purification allowed extensive removal of both impurities. Applying of purified PEG led to 50% lower aggregate fractions. Surprisingly, PEG double purification or addition of methionine prior to crystallization prevented crystal formation. With increased PEG concentration or spiking with peroxides and formaldehydes, crystal formation could be recovered again. With these results, we demonstrate that minimum amounts of oxidizing impurities and thus in consequence chemically altered proteins are vital to initiate mAb1 crystallization. The present study calls PEG as good precipitant for therapeutic biopharmaceuticals into question.

Quality control of protein crystal suspensions using microflow imaging and flow cytometry.

Protein crystallization is an attractive method for protein processing and formulation. However, minor changes in the crystallization setup can lead to changes in the crystal structure or the formation of amorphous protein aggregates, which affect the product quality. Only few analytical tools for qualitative and quantitative differentiation between protein crystals and amorphous protein exist. Electron microscopy requires expensive instrumentation, demanding sample preparation, and challenging image analysis. Therefore, there is a need to establish other analytical techniques. It was the aim of this study to investigate the capability of light obscuration (LO), microflow imaging (MFI), and flow cytometry (FC) in differentiating the amorphous and crystalline states of insulin as a relevant model. Qualitative discrimination of the two populations based on the particle size was possible using LO. Quantitative determination of amorphous protein and crystals by MFI was challenging due to overlapping size distributions. This problem was overcome by particle analysis based on the mean light intensity. Additionally, FC was applied as a new method for the determination of the quality and quantity of amorphous protein by differences in the light scattering. Our results show the potential of MFI and FC for rapid high throughput screening of crystallization conditions and product quality.

Database supported candidate search for metabolite identification.

Mass spectrometry is an important analytical technology for the identification of metabolites and small compounds by their exact mass. But dozens or hundreds of different compounds may have a similar mass or even the same molecule formula. Further elucidation requires tandem mass spectrometry, which provides the masses of compound fragments, but in silico fragmentation programs require substantial computational resources if applied to large numbers of candidate structures. We present and evaluate an approach to obtain candidates from a relational database which contains 28 million compounds from PubChem. A training phase associates tandem-MS peaks with corresponding fragment structures. For the candidate search, the peaks in a query spectrum are translated to fragment structures, and the candidates are retrieved and sorted by the number of matching fragment structures. In the cross validation the evaluation of the relative ranking positions (RRP) using different sizes of training sets confirms that a larger coverage of training data improves the average RRP from 0.65 to 0.72. Our approach allows downstream algorithms to process candidates in order of importance.