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

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

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

Process Understanding in Freeze-Drying Cycle Development: Applications for Through-Vial Impedance Spectroscopy (TVIS) in Mini-pilot Studies

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The Influence of Moisture Content and Temperature on the Long-Term Storage Stability of Freeze-Dried High Concentration Immunoglobulin G (IgG)

High protein concentration products for targeted therapeutic use are often freeze-dried to enhance stability. The long-term storage stability of freeze-dried (FD) plasma-derived Immunoglobulin G (IgG) from moderate to high concentrations (10–200 mg/mL) was assessed. Monomer content, binding activity and reconstitution times were evaluated over a 12-month period under accelerated and real-term storage conditions. In the first case study it was shown that FD IgG from 10 to 200 mg/mL had minimal monomer/activity losses at up to ambient temperature after 12 months of storage. However, at 45 °C the sucrose-to-protein ratio played a significant impact on IgG stability above 50 mg/mL. All IgG concentrations witnessed moisture ingress over a 12-month period. The impact of moisture ingress from environmental exposure (between 0.1% and 5% w/w moisture) for IgG 50 mg/mL was assessed, being generated by exposing low moisture batches to an atmospheric environment for fixed time periods. Results showed that at −20 °C and 20 °C there was no significant difference in terms of monomer or antigen-binding activity losses over 6 months. However, at 45 °C, there were losses in monomer content, seemingly worse for higher moisture content samples although model binding activity indicated no losses. Finally, the difference between a low moisture product (0.1–1% w/w) and a moderately high moisture (3% w/w) product generated by alternative freeze-drying cycles, both stoppered under low oxygen headspace conditions, was evaluated. Results showed that at −20 °C and 20 °C there was no difference in terms of binding activity or monomer content. However, at 45 °C, the low moisture samples had greater monomer and binding activity losses than samples from the highest moisture cycle batch, indicating that over-drying can be an issue

Investigating Alternative Container Formats for Lyophilization of Biological Materials Using Diphtheria Antitoxin Monoclonal Antibody as a Model Molecule

When preparing biological reference materials, the stability of the lyophilized product is critical for long-term storage, particularly in order to meet WHO International Standards, which are not assigned expiry dates but are expected to be in use for several decades. Glass ampoules are typically used by the National Institute for Biological Standards and Control (NIBSC) for the lyophilization of biological materials. More recently, a clear need has arisen for the filling of smaller volumes, for which ampoules may not be optimal. We investigated the use of plastic microtubes as an alternative container for small volume fills. In this study, a recombinant diphtheria antitoxin monoclonal antibody (DATMAB) was used as a model molecule to investigate the suitability of plastic microtubes for filling small volumes. The stability and quality of the dried material was assessed after an accelerated degradation study using a toxin neutralization test and size exclusion HPLC. While microtubes have shown some promise in the past for use in the lyophilization of some biological materials, issues with stability may arise when more labile materials are freeze-dried. We demonstrate here that the microtube format is unsuitable for ensuring the stability of this monoclonal antibody

Freeze-drying cycle optimization for the rapid preservation of protein-loaded liposomal formulations

Technology such as the use of microfluidics to generate liposomes has been well researched, yet the stabilisation of liposomal formulations is a major challenge to their greater implementation. To the best of our knowledge, this is the first study investigating the use of 96 well plates to freeze-dry ovalbumin (OVA) loaded neutral (DMPC:Chol and DSPC:Chol), anionic (DSPC:Chol:PS) and cationic (DSPC:Chol:DOTAP) liposomes. Through the use of high throughput screening, a freeze drying cycle was optimised; ramp freezing from from 4°C to -45°C, followed by primary drying at -30°C and secondary drying at 30°C under a vacuum of 0.1 mBar. These parameters maintained liposome physicochemical properties, with the liposomes remaining below 100 nm and were homogenous (polydispersity index of less than 0.2 post rehydration). Minimal leakage of the OVA protein was observed, with almost 100% OVA remaining encapsulated post rehydration of the formulations. Here we have identified a simple method that allows for the rapid screening and freeze-drying of a range of liposomal formulations