Morphological Properties of Methacrylate-Based Polymer Monoliths: From Gel Porosity to Macroscopic Inhomogeneities.

Shaping chemical interfaces of hard and soft matter materials into physical morphologies that guarantee excellent transport properties is of central importance for technologies relying on adsorption, separation, and reaction at the interface. Polymer monoliths with a hierarchically structured pore space, for example, are widely used in flow-driven processes, whose efficiency depends on the morphology of the support material over several length scales. Compared with alternative support structures, particularly silica monoliths, polymer monoliths yield lower efficiency, which suggests a suboptimal morphology. Based on physical reconstruction by serial block-face scanning electron microscopy we evaluate the structural features of a methacrylate-based polymer monolith from the pore scale to the column scale. The morphological data reveal a homogeneous polymer skeleton with a solute-impenetrable core-porous shell architecture and a heterogeneous macropore space that suffers from inhomogeneities at the short-range and the transcolumn scale. Although the morphology of the polymer phase is favorable to efficient mass transport, the performance of the polymer monolith is limited by severe transcolumn gradients in macroporosity and macropore size. We propose to overcome these morphological limitations by pursuing a preparation strategy that involves active rather than passive shaping of the macropore space, for example, by using silica monoliths as templating structures for polymer monolith preparation.

High throughput multidimensional liquid chromatography approach for online protein removal and characterization of polysorbates and poloxamer in monoclonal antibody formulations.

The majority of commercially available monoclonal antibody (mAb) formulations are stabilized with one of three non-ionic surfactants: polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer 188 (P188). All three surfactants are susceptible to degradation, which can result in functionality loss and subsequent protein aggregation or free fatty acid particle formation. Consequently, quantitative, and qualitative analysis of surfactants is an integral part of formulation development, stability, and batch release testing. Due to the heterogeneous nature of both polysorbates and poloxamer, online isolation of all the compounds from the protein and other excipients that may disturb the subsequent liquid chromatography with charged aerosol detection (LC-CAD) analysis poses a challenge. Herein, we present an analytical method employing LC-CAD, utilizing a combination of anion and cation exchange columns to completely remove proteins online before infusing the isolated surfactant onto a reversed-phase column. The method allows high throughput analysis of polysorbates within 8 minutes and poloxamer 188 within 12 minutes, providing a separation of the surfactant species of polysorbates (unesterified species, lower esters, and higher esters) and poloxamer 188 (early eluters and main species). Accuracy and precision assessed according to the International Council for harmonisation (ICH) guideline were 96 - 109 % and ≤1 % relative standard deviation respectively for all three surfactants in samples containing up to 110 mg/mL mAb. Subsequently, the method was effectively applied to quantify polysorbate 20 and polysorbate 80 in nine commercial drug products with mAb concentration of up to 180 mg/mL.

Degradation of polysorbate investigated by a high-performance liquid chromatography multi-detector system with charged aerosol and mass detection.

Polysorbate 80 is widely used as a formulation component in biopharmaceutical drug products. Recent studies have shown that polysorbate 80 is readily degraded either through direct or indirect means. The degradation of polysorbate 80 causes a concern for the long-term stability of biopharmaceutical drug product, as the breakdown products of polysorbate 80 have been shown to cause adverse effects, such as formation of sub-visible and visible particles and mAb aggregation. Understanding the path and extent of degradation is of a paramount importance for the formulator during formulation development. A multi-detector HPLC system using charged aerosol and mass detection was developed and optimized for the characterization of polysorbate 80 standards. The system included a post-column make-up flow, i.e. an inverse gradient, that enabled constant eluent composition at the detectors. The inverse gradient eliminated the main source of variability for the charged aerosol detector response, thereby enabling the calculation of the mass balance between polysorbate components with different degrees of esterification. Extracted ion chromatograms of the mass detector combined with their respective retention times were used to qualitatively characterize the polysorbate samples down to the individual components. The system was applied to study the degradation of several polysorbate standards which occurred by enzymatic digestion or long-term storage. The system provided detailed information on the mechanism of degradation without the need for additional orthogonal analytical techniques.

Reconstruction and characterization of a polymer-based monolithic stationary phase using serial block-face scanning electron microscopy.

Porous, polymer-based materials are increasingly used as stationary phases in separation science and catalysis, yet their morphology remains largely unknown. The main difficulty lies in reconciling their soft matter nature with the demands of microscopic imaging techniques. We analyze the morphology of a hyper-cross-linked poly(styrene-divinylbenzene) monolith in capillary column format from a sample volume of 60.5 × 60.5 × 19.9 µm(3) reconstructed by serial block-face scanning electron microscopy. To obtain a suitable specimen, the polymer skeleton was stained with tetraphenyllead and the void space filled with resin before the whole monolith was resin-embedded after removing the fused-silica capillary. Chord length distribution analysis revealed characteristic lengths of 7.32 and 0.73 µm, corresponding to two distinct macropore types. The macroporosity (77% on average) was found to increase systematically from the wall to the center. Our results provide valuable insights into the formation process of the monolith and its stationary-phase properties.

Confocal laser scanning microscopy method for quantitative characterization of silica monolith morphology.

We present a fast, nondestructive, and quantitative approach to characterize the morphology of capillary silica-based monolithic columns by reconstruction from confocal laser scanning microscopy images. The method comprises column pretreatment, image acquisition, image processing, and statistical analysis of the image data. The received morphological data are chord length distributions for the bulk macropore space and skeleton of the silica monolith. The morphological information is shown to be comparable to that derived from transmission electron microscopy, but far easier to access. The approach is generally applicable to silica-based capillary columns, monolithic or particulate. It allows the rapid acquisition of hundreds of longitudinal and cross-sectional images in a single session, resolving a multitude of morphological details in the column.

Assessing structural correlations and heterogeneity length scales in functional porous polymers from physical reconstructions.

A general, model-free, quantitative approach to the key morphological properties of a porous polymer monolith is presented. After 3D reconstruction, image-based analysis delivers detailed spatial and spatially correlated information on the structural heterogeneities in the void space and the polymer skeleton. Identified heterogeneities, which limit the monolith's performance in targeted applications, are traced back to the preparation process.

Morphology and separation efficiency of a new generation of analytical silica monoliths.

The heterogeneous morphology of current silica monoliths hinders this column type to reach its envisioned performance goals. We present a new generation of analytical silica monoliths that deliver a substantially improved separation efficiency achieved through several advances in monolith morphology. Analytical silica monoliths from the 1st and 2nd Chromolith generation are characterized and compared by chromatographic methods, mercury intrusion porosimetry, scanning electron microscopy, and confocal laser scanning microscopy. The latter method is instrumental to quantify morphological differences between the monolith generations and to probe the radial variation of morphological properties. Compared with the 1st generation, the new monoliths possess not only smaller macropores, a more homogeneous macropore space, and a thinner silica skeleton, but also radial homogeneity of these structural parameters as well as of the local external or macroporosity. The 66.5% reduction in minimum plate height observed between silica monoliths of the 1st and 2nd Chromolith generation can thus be attributed to two key improvements: a smaller domain size at simultaneously increased macropore homogeneity and the absence of radial morphology gradients, which are behind the considerable peak asymmetry of the 1st generation.