摘要 :
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 prior...
展开
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 ( T _(m)) 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 T _(m) of G-CSF.
收起
摘要 :
Protein engineering and formulation optimization strategies can be taken to minimize protein aggregation in the biopharmaceutical industry. Short-term stability measures such as the midpoint transition temperature ( T _(m)) for gl...
展开
Protein engineering and formulation optimization strategies can be taken to minimize protein aggregation in the biopharmaceutical industry. Short-term stability measures such as the midpoint transition temperature ( T _(m)) for global unfolding provide convenient surrogates for longer-term (e.g., 2-year) degradation kinetics, with which to optimize formulations on practical time-scales. While successful in some cases, their limitations have not been fully evaluated or understood. T _(m) 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 T _(m) of an antibody Fab fragment only correlated with its rate of monomer loss at temperatures close to the T _(m). Here we evaluated T _(m), the fraction of unfolded protein ( f _( T )) 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 T _(m) between each pair of GCSF variants increased. The rank order of formulations determined by T _(m) was the best preserved, with R ~(2)-values >0.7. T _(m)-values also provided a good predictor ( R ~(2) = 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 ( f _(T37)), but transitioned smoothly to a constant baseline rate of aggregation at f _(T37) < 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 destabilized 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.
收起
摘要 :
Formulation is critical to successful delivery of lyophilized biologics. We have compared the impact of buffer choice and the addition of sodium chloride (a formulant often viewed as unfavorable for freeze-drying applications) on ...
展开
Formulation is critical to successful delivery of lyophilized biologics. We have compared the impact of buffer choice and the addition of sodium chloride (a formulant often viewed as unfavorable for freeze-drying applications) on the outcome of trial lyophilization of an interleukin-6 reference material. While phosphate buffer was a preferred choice and yielded well-formed cakes associated with fair recovery of biological activity, the resultant residual moisture content was high (2–4% w/w). By inclusion of isotonic levels of NaCl, the freeze-dried appearance and process were not impaired, but the residual moisture delivered was considerably reduced to levels <1% w/w. We postulate that this is due to the presence of a more open-cake structure and support this with evidence from thermal analysis and scanning electron microscopy. This work illustrates the importance of wide ranging empirical investigation of formulation options in order to optimize freeze-drying outcomes for biologics.
收起
摘要 :
Biosimilar medicines are having a global effect, and as such regulatory agencies worldwide are assessing how to respond to them. Here, two events held by GaBI for the Colombian medicines regulatory agency (INVIMA) are reported. Th...
展开
Biosimilar medicines are having a global effect, and as such regulatory agencies worldwide are assessing how to respond to them. Here, two events held by GaBI for the Colombian medicines regulatory agency (INVIMA) are reported. The first was an educational workshop held in 2016 to discuss approaches to evaluating biosimilar products and the second was a scientific meeting on the quality assessment for biosimilars held in 2017. Both provided a forum to exchange knowledge on best practice in this new and important area.
收起
摘要 :
The lyophilization of proteins in microplates, to assess and optimise formulations rapidly, has been applied for the first time to a therapeutic protein and, in particular, one that requires a cell-based biological assay, in order...
展开
The lyophilization of proteins in microplates, to assess and optimise formulations rapidly, has been applied for the first time to a therapeutic protein and, in particular, one that requires a cell-based biological assay, in order to demonstrate the broader usefulness of the approach. Factorial design of experiment methods were combined with lyophilization in microplates to identify optimum formulations that stabilised granulocyte colony-stimulating factor during freeze drying. An initial screen rapidly identified key excipients and potential interactions, which was then followed by a central composite face designed optimisation experiment. Human serum albumin and Tween 20 had significant effects on maintaining protein stability. As previously, the optimum formulation was then freeze-dried in stoppered vials to verify that the microscale data is relevant to pilot scales. However, to validate the approach further, the selected formulation was also assessed for solid-state shelf-life through the use of accelerated stability studies. This approach allows for a high-throughput assessment of excipient options early on in product development, while also reducing costs in terms of time and quantity of materials required.
收起
摘要 :
Residual moisture is a key quality control parameter for lyophilized biologicals, as high moisture can correlate with poor stability. Coulometric Karl Fischer titration is the most widely used technology to determine residual wate...
展开
Residual moisture is a key quality control parameter for lyophilized biologicals, as high moisture can correlate with poor stability. Coulometric Karl Fischer titration is the most widely used technology to determine residual water content; some chemicals are known to cause problems with Karl Fischer titration, but these chemicals do not usually occur in biologics. Three biological samples, of fibrinogen, heparin and Haemophilus influenza b polysaccharide, have caused particular issue in our hands by routine Karl Fischer analysis, illustrating different limitations with this method. The use of thermogravimetric analysis, with evolved gas mass spectrometric monitoring, is described here as a successful alternative for moisture analysis in these materials.
收起
摘要 :
Biologicals cover a wide range of materials which are often labile in solution and require lyophilization to stabilize I them. For instance Factor VIII activity begins to fall as soon as a sample of plasma is taken and whether a p...
展开
Biologicals cover a wide range of materials which are often labile in solution and require lyophilization to stabilize I them. For instance Factor VIII activity begins to fall as soon as a sample of plasma is taken and whether a purified concentrate from plasma or a recombinant product of DNA technology is considered, both show loss of activity during processing and on storage and so one of the challenges of formulation is to stabilize the biological material for the shelf life of the product [1]. In order to do this water is removed by lyophilization and this usually reduces the degradation rate significantly. Towns [2] reviewed the interaction between water and proteins; stripping the water to below a monolayer covering of the surface of the protein generally engenders greater dry-state stability. Higher water content in lyophilized material plasticises the protein and allows for greater mobility as well as lowering the glass transition temperature, which can impact stability however formulation must be optimized so as to protect the dehydrated biological from damage [3].
收起
摘要 :
Residual moisture content and formulation are important parameters when preparing lyophilized reference materials containing labile proteins. The protection of Factor VIII and Factor V activities were monitored in a lyophilized pl...
展开
Residual moisture content and formulation are important parameters when preparing lyophilized reference materials containing labile proteins. The protection of Factor VIII and Factor V activities were monitored in a lyophilized plasma preparation following formulation with either no additional excipient, 40 mM Hepes (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 10 mg/mL glycine or a combination of 40 mM Hepes and 10 mg/mL glycine. The preservation of Factor VIII activity during freeze-drying was improved by the addition of either stabiliser and improved most, amongst the options studied, by the addition of both glycine and Hepes. The predicted stability at ?20 °C and 20 °C was estimated using accelerated degradation studies. Although for plasma lyophilized alone there was some benefit from further desiccation over phosphorus pentoxide, resulting in very low moistures, for suitably formulated samples the predicted stability was as good for freeze-dried only samples as for those with further desiccation. This study emphasises the importance of optimum formulation on the stability of lyophilized proteins.
收起
原文获取