Asymmetrical flow field-flow-fractionation in pharmaceutical analytics

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.