65 research outputs found
Structure and ligand-binding properties of abnormal human albumins
Albumins Redhill, Warwick-1 and Carlisle are
monomeric slow albumin variants discovered in sera
obtained from patients in unrelated families resident
in the U.K.
Albumin Redhill had previously been studied in this
laboratory and was here purified by fast protein liquid
chromatrography (FPLC) and was subsequently submitted for
amino acid sequencing by the solid-phase Edman process.
It was found that Albumin Redhill has an extra N-terminal
arginine residue, and this places it into a new class
of albumin variants.
The binding of nickel and copper was studied in
greater depth than previously, and the binding of these
metal ions at the primary N-terminal site confirmed these
as being significantly inhibited due to the inclusion of
the extra basic amino acid residue. The binding of warfarin
to Albumin Redhill is reduced compared to normal albumin.
Albumin Carlisle, a heat-stable variant, was found
in three members of a family of English origin from
Carlisle. The binding of a range of dyes and the
electrophoretic mobility on a series of media were assessed.
The variant Albumin Carlisle was purified to homogeneity
by FPLC chromatofocusing and was shown to be antigenically
indistinguishable from albumin A, although it does have
a more basic isoelectric point (5.74 compared to 5.63 for
normal albumin). The evidence from both electrophoretic
and chromatographic procedures are consistent with an
acid
+
neutral amino acid mutation, and studies of the
CNBr fragments of the variant suggest that the site of
mutation is in the region 329-548 residues.
Reverse-phase HPLC has been used to pinpoint a
difference in the profile of the tryptic digest of the
variant albumin from the normal, and it may be that this
technique could be utilised to obtain molecular data on
the mutation.
The ligand binding properties of metal ions,
bilirubin, palmitate and warfarin were assessed and it
was shown that Albumin Carlisle has increased warfarin
binding but decreased bilirubin affinity, although
the binding of metal ions and palmitate was unaffected
The importance of formulation in the successful lyophilization of influenza reference materials.
© 2014.Lyophilized Influenza antigen reference reagents are a critical resource in the quality control of influenza vaccines. A standard formulation has been used successfully at NIBSC for many years however, following the unexpected occurrence of a collapsed appearance in a particular batch a study was carried out to establish the impact of the sugar concentration in the formulation using modulated differential scanning calorimetry (mDSC) and nuclear magnetic resonance spectroscopy (NMR).There was a correlation between the presence and size of the mDSC eutectic temperature events and the freeze dried appearance of the cakes, which became progressively worse with increasing amounts of sugar. NMR spectroscopy could be used to positively identify and quantify the sugars in the formulations. MDSC can rapidly predict if the freeze dried appearance will be acceptable so as to assure the successful lyophilization of influenza reference preparations
HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions
Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein–excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients. These interactions were related to their influence on the thermal-melting temperatures (Tm) for the nonreversible unfolding of G-CSF in liquid formulations. The residue-level interaction sites predicted in silico correlated well with those identified experimentally and highlighted the potential impact of specific excipient interactions on the Tm of G-CSF
Vacuum-Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How Does Nucleation Control Affect Protein Stability?
Abstract In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. In addition, the effect of 2 different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study
Freeze-drying microscopy unravelling the complexities of freeze-drying pharmaceuticals with advanced microscopy techniques
Degradation of stored material, either through autolysis or the growth of spoilage organisms, is primarily dependent on the presence of water. Products that are prone to degradation, such as food and pharmaceuticals, must be stabilized by immobilizing or reducing of the water content. For example, vaccines and other biological materials can be stabilized by chilling or freezing, but transporting samples in a frozen state is expensive, and breakdown of freezers may result in the complete loss of valuable product. Alternatively, water can be removed from labile products through air-drying using high processing temperatures, but this can alter the product's physical and chemical properties and is therefore unsuitable for pharmaceuticals
Impact of controlled vacuum induced surface freezing on the freeze drying of human plasma
During the freezing step of a typical freeze drying process, the temperature at which nucleation is induced is generally stochastically distributed, resulting in undesired within-batch heterogeneity. Controlled nucleation techniques have been developed to address this problem; these make it possible to trigger the formation of ice crystals at the same time and temperature in all the batch. Here, the controlled nucleation technique known as vacuum induced surface freezing is compared to spontaneous freezing for the freeze drying of human plasma, a highly concentrated system commonly stored in a dried state. The potency of Factor VIII (FVIII), a sensitive, labile protein present in plasma, and the reconstitution time of the dried cakes are evaluated immediately after freeze drying, and after 1, 3, 6 or 9 months storage at different degradation temperatures. We show that the application of controlled nucleation significantly reduces the reconstitution time and in addition helps to improve FVIII stability
Tm-values and unfolded fraction can predict aggregation rates for GCSF variant formulations, but not under predominantly native conditions.
Protein engineering and formulation optimisation strategies can be taken to minimise protein aggregation in the biopharmaceutical industry. Short-term stability measures such as the mid-point transition temperature (Tm) for global unfolding provide convenient surrogates for longer-term (eg 2-year) degradation kinetics, with which to optimise formulations on practical time-scales. While successful in some cases, their limitations have not been fully evaluated or understood. Tm values are known to correlate with chemical degradation kinetics for wild-type granulocyte colony stimulating factor (GCSF) at pH 4-5.5. However, we found previously that the Tm of an antibody Fab fragment, only correlated with its rate of monomer loss at temperatures close to the Tm. Here we evaluated Tm, the fraction of unfolded protein (fT) at temperature T, and two additional short-term stability measures, for their ability to predict the kinetics of monomer and bioactivity loss of wild-type GCSF and four variants, at 37 °C, and in a wide range of formulations. The GCSF variants introduced one to three mutations, giving a range of conformational stabilities spanning 7.8 kcal mol-1. We determined the extent to which the formulation rank order differs across the variants, when evaluated by each of the four short-term stability measures. All correlations decreased as the difference in average Tm between each pair of GCSF variants increased. The rank order of formulations determined by Tm was the best preserved, with R2-values >0.7. Tm-values also provided a good predictor (R2 = 0.73) of the aggregation rates, extending previous findings to include GCSF variant-formulation combinations. Further analysis revealed that GCSF aggregation rates at 37 °C, were dependent on the fraction unfolded at 37 °C (fT37), but transitioned smoothly to a constant baseline rate of aggregation at fT37 <10-3. A similar function was observed previously for A33 Fab formulated by pH, ionic strength and temperature, without excipients. For GCSF, all combinations of variants and formulations fit onto a single curve, suggesting that even single mutations destabilised by up to 4.8 kcal mol-1, are insufficient to change significantly the baseline rate of aggregation under native conditions. The baseline rate of aggregation for GCSF under native conditions, was 66-fold higher than that for A33 Fab, highlighting that they are a specific feature of each native protein structure, likely to be dependent on local surface properties and dynamics
Process Understanding in Freeze-Drying Cycle Development: Applications for Through-Vial Impedance Spectroscopy (TVIS) in Mini-pilot Studies
The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link
NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF.
Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, molded by evolution of the molecule. Identifying which protein amino acid residues jeopardize global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Here, we use 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte colony-stimulating factor (G-CSF) through thermal perturbation. From this analysis, we identified four residues (G4, A6, T133, and Q134) that we classed as significant to global stability, given that they all experienced large environmental and dynamic changes and were closely correlated to each other in their NMR characteristics. Additionally, we observe that roughly four structural clusters are subject to localized conformational changes or partial unfolding prior to global unfolding at higher temperature. Combining NMR observables with structure relaxation methods reveals that these structural clusters concentrate around loop AB (binding site III inclusive). This loop has been previously implicated in conformational changes that result in an aggregation prone state of G-CSF. Residues H43, V48, and S63 appear to be pivotal to an opening motion of loop AB, a change that is possibly also important for function. Hence, we present here an approach to profiling residues in order to highlight their potential roles in the two vital characteristics of proteins: stability and bioactivity
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