Comparison of Submicron Particle Counting Methods with a Heat Stressed Monoclonal Antibody: Effect of Electrolytes and Implications on Sample Preparation.

Within this study, the performance and limitations of tunable resistive pulse sensing (TRPS) was evaluated to characterize submicron particles in unstressed and heat stressed monoclonal antibody (mAb) solutions. These were compared with microfluidic resistive pulse sensing (MRPS), resonant mass measurement (RMM), and nanoparticle tracking analysis (NTA). For TRPS and MRPS measurements, an adjustment of ionic strength was required to achieve suitable measurement conditions. The addition of electrolytes is potentially critical for protein formulations and therefore the effect of salt concentration and pH on submicron particle levels was further investigated. Heat stress caused a sharp increase in particle levels between 250-900 nm, observable by all four techniques. Due to reduced colloidal stability, indicated by increased attractive forces and reduced aggregation onset temperatures in the presence of sodium chloride, protein aggregation was observed in heat stressed mAb only after the addition of sodium chloride. Achieving adequate ionic strength by replacing sodium chloride with other electrolytes similarly resulted in reduced colloidal stability and protein aggregation. It is recommended that protein samples prone for aggregation in the presence of high ionic strength should not be analyzed by RPS measurements after the addition of electrolytes. However, protein samples containing already required ionic strength can be analyzed by any of the four techniques.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Simultaneous automated image analysis and Raman spectroscopy of powders at an individual particle level.

Solid form diversity of raw materials can be critical for the performance of the final drug product. In this study, Raman spectroscopy, image analysis and combined Raman and image analysis were utilized to characterize the solid form composition of a particulate raw material. Raman spectroscopy provides chemical information and is complementary to the physical information provided by image analysis. To demonstrate this approach, binary mixtures of two solid forms of carbamazepine with a distinct shape, an anhydrate (prism shaped) and a dihydrate (needle shaped), were characterized at an individual particle level. Partial least squares discriminant analysis classification models were developed and tested with known, gravimetrically mixed test samples, followed by analysis of unknown, commercially supplied carbamazepine raw material samples. Classification of several thousands of particles was performed, and it was observed that with the known binary mixtures, the minimum number of particles needed for the combined Raman spectroscopy - image analysis classification model was approximately 100 particles per solid form. The carbamazepine anhydrate and dihydrate particles were detected and classified with a classification error of 1 % using the combined model. Further, this approach allowed the identification of raw material solid form impurity in unknown raw material samples. Simultaneous automated image analysis and Raman spectroscopy of powders at an individual particle level has its potential in accurate detection of low amounts of unwanted solid forms in particulate raw material samples.

Immunological Evaluation In Vitro of Nanoparticulate Impurities Isolated From Pharmaceutical-Grade Sucrose.

Sucrose is a commonly used stabilizing excipient in protein formulations. However, recent studies have indicated the presence of nanoparticulate impurities (NPIs) in the size range of 100-200 nm in pharmaceutical-grade sucrose. Furthermore, isolated NPIs have been shown to induce protein aggregation when added to monoclonal antibody formulations. Moreover, nanoparticles are popular vaccine delivery systems used to increase the immunogenicity of antigens. Therefore, we hypothesized that NPIs may have immunostimulatory properties. In this study, we evaluated the immunomodulatory effects of NPIs in presence and absence of trastuzumab in vitro with monocyte-derived dendritic cells (moDCs). Exposure of trastuzumab, the model IgG used in this study, to NPIs led to an increase in concentration of proteinaceous particles in the sub-micron range. When added to moDCs, the NPIs alone or in presence of trastuzumab did not affect cell viability or cytotoxicity. Moreover, no significant effect on the expression of surface markers, and cytokine and chemokine production was observed. Our findings showed, surprisingly, no evidence of any immunomodulatory activity of NPIs. As this study was limited to a single IgG formulation and to in vitro immunological read-outs, further work is required to fully understand the immunogenic potential of NPIs.

What Makes Polysorbate Functional? Impact of Polysorbate 80 Grade and Quality on IgG Stability During Mechanical Stress.

Polysorbate 80 (PS80) is a commonly used surfactant in therapeutic protein formulations to mitigate adsorption and interface-induced protein aggregation. Several PS80 grades and qualities are available on the market for parenteral application. The role of PS80 grade on protein stability remains debatable, and the impact of (partially) degraded PS on protein aggregation is not yet well understood. In our study, a monoclonal antibody (IgG) was subjected to 3 different mechanical stress conditions in the presence of multicompendial (MC) and Chinese pharmacopeia (ChP) grade PS80. Furthermore, IgG formulations were spiked with (partly) hydrolyzed PS80 to investigate the effect of PS80 degradants on protein stability. PS80 functionality was assessed by measuring the extent of protein aggregation and particle formation induced during mechanical stress by using size-exclusion chromatography, dynamic light scattering, backgrounded membrane imaging, and flow imaging microscopy. No distinguishable differences in PS80 functionality between MC and ChP grade were observed in the 3 stress tests. However, with increasing degree of PS80 hydrolysis, higher counts of subvisible particles were measured after stress. Furthermore, higher levels of PS80 degradants at a constant PS80 concentration may destabilize the IgG. In conclusion, MC and ChP grade PS80 are equally protective, but PS80 degradants compromise IgG stability.

Critical Evaluation of Microfluidic Resistive Pulse Sensing for Quantification and Sizing of Nanometer- and Micrometer-Sized Particles in Biopharmaceutical Products.

The objective was to evaluate performance, strengths, and limitations of the microfluidic resistive pulse sensing (MRPS) technique for the characterization of particles in the size range from about 50 to 2000 nm. MRPS, resonant mass measurement (RMM), nanoparticle tracking analysis (NTA) and dynamic light scattering were compared for the analysis of nanometer-sized polystyrene (PS) beads, liposomes, bacteria, and protein aggregates. An electrical conductivity of at least 3 mS/cm (equivalent to 25 mM NaCl) was determined as a key requirement for reliable analysis with MRPS. Particle size distributions of PS beads determined by MRPS, NTA, and RMM correlated well. However, counting precision varied significantly among the techniques and was best for RMM followed by MRPS and NTA. As determined by measuring single and mixed PS bead populations, MRPS showed the highest peak resolution for sizing. RMM and MRPS were superior over dynamic light scattering and NTA for the characterization of stressed protein samples. Finally, MRPS proved to be the only analytical technique able to characterize both bacteria and liposomes. In conclusion, MRPS is an orthogonal technique alongside other established techniques for a comprehensive analysis of a samples particle size distribution and particle concentration.

Inhalable spray-dried formulation of D-LAK antimicrobial peptides targeting tuberculosis.

Tuberculosis (TB) is a global disease that is becoming more difficult to treat due to the emergence of multidrug resistant (MDR) Mycobacterium tuberculosis. Inhalable antimicrobial peptides (AMPs) are potentially useful alternative anti-TB agents because they can overcome resistance against classical antibiotics, reduce systemic adverse effects, and achieve local targeting. The aims of the current study were to produce inhalable dry powders containing d-enantiomeric AMPs (D-LAK120-HP13 and D-LAK120-A) and evaluate their solid state properties, aerosol performance, and structural conformation. These two peptides were spray dried with mannitol as a bulking agent at three mass ratios (peptide:mannitol 1:99, 1:49, and 1:24) from aqueous solutions. The resultant particles were spherical, with those containing D-LAK120-HP13 being more corrugated than those with D-LAK120-A. The median volumetric diameter of the particles was approximately 3µm. The residual water content of all powders were <3% w/w and crystalline, due to the low hygroscopicity and crystallinity of mannitol, respectively. The mannitol changed from a mixture of alpha- and beta-forms to delta form with an increasing proportion of AMP in the formulation. The emitted fraction and fine particle fraction of the powders when dispersed from an Osmohaler(®) at 90L/min were about 80% and 50-60% of the loaded dose, respectively, indicating good aerosol performance. Circular dichroism data showed that D-LAK120-HP13 dissolved in Tris buffer at pH 7.15 was of a disordered conformation. In contrast, D-LAK120-A showed greater α-helical conformation. Since the conformations of the AMPs were comparable to the controls (unprocessed peptides), the spray drying process did not substantially affect their secondary structures. In conclusion, spray dried powders containing d-enantiomeric AMPs with preserved secondary molecular structures and good aerosol performance could be successfully produced. They may potentially be used for treating MDR-TB when delivered by inhalation.