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Fraunhofer, Wolfgang (2003): Asymmetrical flow field-flow-fractionation in pharmaceutical analytics: Investigations in aggregation tendencies of pharmaceutical antibodies. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Due to enormous progress in recombinant DNA techniques and methodology, a multitude of biosynthetic, pharmaceutically relevant polypeptides and proteins became available in the past decade and have been employed in numerous pharmaceutical products. Concomitantly, substantial progress was made in pharmaceutical formulation development of peptides and proteins, inasmuch as many challenges in formulating these compounds in products with optimal therapeutic effects and shelf life were successfully approached. Additionally, new drug delivery systems – e.g., based on polymeric materials – will most likely enlarge the spectrum of future proteinic dosage forms, where today solutions and lyophilized products take center stage. Yet, due to the proneness of proteins to degradation - what can affect pharmaceutical relevant features such as biological activity and immunogenicity -, scrutinizing the homogeneity of protein formulations is of utmost importance. Hence, the development and implementation of new analytical techniques in order to keep pace is highly desired. It was the aim of this thesis to evaluate the applicability of asymmetrical flow field flow fractionation (AF4) in pharmaceutical protein analytics, to compare AF4 performance with established state-of-the-art methods and to reveal the effectivity of inherent AF4 characteristics in demanding analytical tasks. The Theoretical Section encompasses Chapter 2, wherein the family of field-flow fractionation techniques is introduced, as well as Chapter 3 (attending to protein aggregation) and Chapter 4, which provides an insight into multi-angle light scattering. The Theoretical Section is summarized in Chapter 5. Chapter 6 attends to the general applicability of (semi-)chromatographic AF4 in protein analysis. The correlation of cross flow progression with increased resolution was exemplified by the separation of human serum albumin (HSA), thereby rendering the (base-line) separation of HSA specimen into monomer, dimer, trimer and tetramer possible. Due to the AF4 feature to discretionarily alter the resolution power within one separation run, the fractionation of higherorder aggregates and insoluble, precipitated protein was successfully performed. System variables and parameters of fractionation were investigated, revealing that sample loads differing more than two orders of magnitude did not negatively affect data reproducibility. Whereas up to now cross flow intensity was deemed to predominantly account for contingent sample loss during AF4 experiments, analysis of proteins with varying hydrophilicity proved the preceding focusing step to contribute notably for that phenomenon. How to overcome potential drawbacks such as sample-membrane interactions by adequate choice of the ultrafiltration membrane as well as carrier liquid composition was illustrated. Chapter 11. Summary, conclusions and prospective 177 Given the background that effective AF4 fractionations are due to differences in analyte size – i.e., in diffusion coefficients -, the separation of equal-sized proteins is prima facie considered to be impractical. Yet, the retaining impact of sample-membrane interaction was evidenced to decrease the effective diffusion coefficient, resulting in successful fractionations of proteins differing ~1% in size (i.e., G-CSF, 19.6 kDa versus IFN-α2a, 19.4 kDa). In this realm, the normal mode elution order of smaller analytes eluting prior to larger ones was shown to be invertible, exemplified by the elution of a 40 kDa analyte prior to IFN-α2a. AF4 potency in analysis of insoluble high molecular weight (hmw) aggregates was compared with data derived by established methods such as light obscuration and Coulter technique, verifying the competitiveness of AF4. A comparative study of AF4 with size exclusion chromatography (SE-HPLC) unveiled SE-HPLC to inhere higher recovery rates and AF4 to exhibit greater resolution. Coupling both techniques with multi-angle light scattering (MALS) detection systems disclosed SE-HPLC to induce artifacts concerning hmw aggregate quantification. Moreover, in contrast to SE-HPLC, AF4 was capable of seizing undissolved and precipitated protein specimen, thus making AF4 a promising alternative in the analysis of protein pharmaceuticals. AF4´s ability to separate undissolved sample components proved to be an indispensable feature in the analysis of a pharmaceutical protein formulated within siliconized disposable syringes, which was attended to in Chapter 7. During long-term storage, visible particulate matter developed sporadically within the syringe volumes, raising the question of the particles´ origin. Since protein instabilities were not to be accounted for being the particle source – verified by several analytical methods -, silicone oil detachment and subsequent coalescence came into question, as the barrel siliconization process was lacking a final heat curing step. Thus, an AF4 application was developed, intending to separate μm sized silicone oil droplets. The task was approached by analysis of silicone oil emulsions, followed by the fractionation of ultrasoundstressed syringe volumes containing detached and coalesced silicone oil after stress exertion. Unambiguously, detached silicone oil was evidenced by AF4 to account for visible particulate matter in the syringe volumes, corroborated by MALS and refractive index detection as well as light microscopy and syringe frictional drag analysis. Subsequently to artificially induced protein aggregation of particle-containing syringe volumes, AF4 was able to separate silicone oil droplets, protein monomer and aggregates as individual fractions within one single run. Finally, AF4 enabled access to data on protein drug stability and insights into protein adsorption tendencies on coalesced silicone oil specimen – thereby providing valuable data which otherwise would have required a variety of various analytical techniques. Chapter 11. Summary, conclusions and prospective 178 In Chapter 8 the suitability of AF4 in overall-characterization of gelatin nanoparticles was explored. The efficacy of providing hmw gelatin bulk material by various desolvation steps was evaluated by SE-HPLC and AF4. Due to the absence of shear degradation phenomena, AF4 was demonstrated to enable more moderate separation conditions than SE-HPLC, verified by on-line determination of analyte molecular weight via MALS. Gelatin nanoparticles were characterized by means of AF4/MALS with respect to size and size distributions and the data were compared to results of photon correlation spectroscopy (PCS) and scanning electron microscopy (SEM). Because of the precedent separation step via AF4, data derived by MALS revealed a greater veracity than PCS results, where the size assessment of nanoparticles relied on batch experiments. Whereas PCS attributed unloaded and DNA plasmid loaded nanoparticles virtually unimodal size distributions, both AF4/MALS and SEM demonstrated the nanoparticles to span a broad size range. Furthermore, loaded and unloaded nanoparticles were unveiled to exhibit only minimal differences in size, thus providing information on the interplay of nanoparticles and plasmid strands. For the first time, the separation of nanocolloidal drug carrier and designated pharmaceutical payload was established. Additionally to drug carrier characteristics, data on the loading efficacy could be yielded. Furthermore, nanoparticle shelf life stability and extent of potential drug decomplexation could be determined. Bearing in mind colloidal, polymer-based drug delivery carriers gaining increasing importance, that very AF4 application is expected to accommodate the demand for accurate analytics, as the pharmaceutical product can be characterized in both qualitative and quantitative terms. In Chapter 9 a case study of particulate matter analysis of a pharmaceutical antibody solution is presented, wherein individual vials of one production lot developed visible components at random during long-term storage. In order to (a) provide evidence on the presence of the contamination, (b) to attempt particulate entitiy quantification and (c) to elucidate particles´ nature, a multiplicity of analytical techniques were applied, encompassing particle counting (optical inspection, light obscuration, light microscopy), protein characterization techniques (SE-HPLC, polyacrylamide gel electrophoresis, AF4, microcalorimetry) and particle separation techniques (sterile filtration, AF4). Attempts to isolate the particulate components by AF4 or filtration techniques provided no further indications of the particle´s origin. Virtually no alterations in protein characteristics were monitored between contaminated and particle-free vial volumes, respectively. Solely, microcalorimetric data of contaminated vial volumes resembled those of immunoglobulin solutions exposed to heat stress prior to analytics. Consequently, protein instabilities were assumed not to cause the visible contamination. Chapter 11. Summary, conclusions and prospective 179 The topic of liquid protein parenterals, protein instability and particulate matter was completed by presenting a formulation process of an immunoglobulin into a liquid formulation in Chapter 10. Prevalent strategies and mainstream trends of liquid protein formulation were introduced by reviewing latest publications on the issue. Parameters revealing decisive influence on the protein´s long-term stability such as solution pH as well as type and concentration of excipients were evaluated by means of accelerated stability studies at various storage temperatures. Additionally, processing parameters, e.g., freeze/thawing, were assessed evaluating criteria in terms of surfactant and buffer choice. The addition of NaCl was shown to detract from protein stability and to facilitate the formation of particulate matter. Non-deleterious alternatives of salt additives were discovered. On the other hand, the addition of polyols such as mannitol and sorbitol was demonstrated to notably contribute to the immunoglobulin stability. Preferential accumulation at the native state protein was thought to be the mechanism for reducing aggregation phenomena of the protein. Besides, the extent of fragmentation was reduced by polyols, indicating a second pathway of stabilization, which was hypothesized to be hampering of oxidation processes. Due to detailed investigations, a proposal pertaining an optimal formulation could be made in the course of that case study. This thesis has shown that asymmetrical flow field-flow fractionation (AF) can effectively be used to monitor protein stability in a broad variety of pharmaceutical formulations. Especially in the characterization of the most common outcome of physical instability – i.e., protein aggregation – the potential of AF4 has comprehensively been demonstrated. Moreover, AF4 applications and separation tasks within pharmaceutical analytics considered hitherto impractical or at least highly challenging were successfully performed. Facing increasingly complex liquid- or colloidal-based formulations, with this knowledge practice and research in pharmaceutical analytics can take a notable step forward.