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Manning M.C.,Legacy BioDesign LLC | Chou D.K.,Genzyme | Murphy B.M.,Legacy BioDesign LLC | Payne R.W.,Legacy BioDesign LLC | Katayama D.S.,Amylin Pharmaceuticals Inc.
Pharmaceutical Research | Year: 2010

In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability. © 2010 Springer Science+Business Media, LLC.


Ohtake S.,Pfizer | Kita Y.,Keio University | Payne R.,Legacy BioDesign LLC | Manning M.,Legacy BioDesign LLC | Arakawa T.,Alliance Protein Laboratories
Protein and Peptide Letters | Year: 2013

Short peptides are important biopharmaceuticals as agonistic or antagonistic ligands, aggregation inhibitors, and vaccines, as well as in many other applications. They behave differently from globular proteins in solution. Many short peptides are unstructured and tend to aggregate and undergo structural transition in response to changes in solvent environment, including pH, temperature, ionic strength, presence of organic solvents or surfactants, and exposure to lipid membranes. Such structural transitions are often associated with fibril or β-amyloid formation. These structural characteristics of short peptides have drastic impact on their function, immunogenicity, and storage stability. © 2013 Bentham Science Publishers.


Merutka G.,Azelon Pharmaceuticals | Murphy B.M.,Colorado State University | Murphy B.M.,Legacy BioDesign LLC | Payne R.W.,Colorado State University | And 6 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2016

The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide. © 2015 Elsevier B.V. All rights reserved.


PubMed | West Coast BioDesign LLC, Azelon Pharmaceuticals, Legacy BioDesign LLC and Colorado State University
Type: | Journal: European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V | Year: 2016

The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide.


Stockdale G.,Glaxosmithkline | Murphy B.M.,Legacy BioDesign LLC | Murphy B.M.,Colorado State University | D'Antonio J.,North Carolina State University | And 3 more authors.
Journal of Pharmaceutical Sciences | Year: 2015

Comparing higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that changes in solution conditions produced detectable changes in the second-derivative amide I Fourier transform infrared (FTIR) spectra for a variety of model proteins. Those comparisons utilized vector-based approaches, such as spectral overlap and spectral correlation coefficients to quantify differences between spectra. In this study, chemometric analyses of the same data were performed, to classify samples into different groups based on the solution conditions received. The solution conditions were composed of various combinations of temperature, pH, and salt types. At first, principal component analysis (PCA) was used to visually demonstrate that FTIR spectra respond to changes in solution conditions, which, presumably indicates variations in HOS. This observed when samples from the same solution condition form clusters within a PCA score plot. The second approach, called soft independent modeling of class analogy (SIMCA), was conducted to account for the within-class experimental error for the lysozyme spectra. The DModX values, indicative of the distance of each spectra to their respective class models, was found to be a more sensitive quantitative indicator of changes in HOS, when compared with the modified area of overlap algorithm. The SIMCA approach provides a metric to determine whether new observations do, or do not belong to a particular class or group. Thus, SIMCA is the recommended approach when multiple samples from each condition are available. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.


Murphy B.M.,Legacy BioDesign LLC | Murphy B.M.,Colorado State University | D'Antonio J.,North Carolina State University | Manning M.C.,Legacy BioDesign LLC | And 2 more authors.
Current Pharmaceutical Biotechnology | Year: 2015

Demonstrating comparability of secondary structure composition as part of higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that the variability of second derivative amide I Fourier transform infrared (FTIR) spectra were small enough that significant differences in secondary structures could be seen for a variety of model proteins. Those comparisons used spectral overlap and spectral correlation coefficients to quantify spectral differences. However, many of the excipients used in downstream purification process, drug substance, and drug product formulation, such as free amino acids and sugars, can interfere with the absorbance in the amide I region. In this study, analysis of amide II FTIR spectra is shown as an alternative to using spectral data from the amide I region to analyze protein secondary structure to assess their HOS. This research provided spectral overlap and spectral correlation coefficient mathematical approaches for analysis of amide II FTIR spectra to demonstrate comparability of protein secondary structure. Spectral overlap and spectral correlation coefficients results show strong correlations between changes in the second derivative of amide II and amide I FTIR spectra for various model proteins under different conditions, which demonstrate the applicability of using amide II FTIR spectra for the comparability of protein secondary structure. These results indicate that the analysis of the second derivative of amide II FTIR spectra may be used to monitor and demonstrate comparability of protein secondary structure during downstream process and formulation development of protein therapeutics. © 2014, Bentham Science Publishers.


PubMed | Colorado State University and LegacyBioDesign LLC
Type: | Journal: Journal of pharmaceutical sciences | Year: 2016

Buffers comprise an integral component of protein formulations. Not only do they function to regulate shifts in pH, they also can stabilize proteins by a variety of mechanisms. The ability of buffers to stabilize therapeutic proteins whether in liquid formulations, frozen solutions, or the solid state is highlighted in this review. Addition of buffers can result in increased conformational stability of proteins, whether by ligand binding or by an excluded solute mechanism. In addition, they can alter the colloidal stability of proteins and modulate interfacial damage. Buffers can also lead to destabilization of proteins, and the stability of buffers themselves is presented. Furthermore, the potential safety and toxicity issues of buffers are discussed, with a special emphasis on the influence of buffers on the perceived pain upon injection. Finally, the interaction of buffers with other excipients is examined.


Nagarkar R.P.,Glaxosmithkline | Nagarkar R.P.,KBI Biopharma | Murphy B.M.,Legacy BioDesign LLC | Yu X.,Glaxosmithkline | And 2 more authors.
Current Pharmaceutical Biotechnology | Year: 2013

Better understanding of protein higher order structures (HOS) is of major interest to researchers in the field of biotechnology and biopharmaceutics. Monitoring a protein's HOS is crucial towards understanding the impact of molecular conformation on the biotechnological application. In addition, maintaining the HOS is critical for achieving robust processes and developing stable formulations of therapeutic proteins. Loss of HOS contributes to increased aggregation, enhanced immunogenicity and loss of function. Selecting the proper biophysical methods to monitor the secondary and tertiary structures of therapeutic proteins remains the central question in this field. In this study, both Fourier Transform Infrared (FTIR) and vibrational circular dichroism (VCD) spectroscopy are employed to characterize the secondary structures of various proteins as a function of temperature and pH. Three proteins with different secondary structures were examined, human serum albumin (HSA), myoglobin, and the monoclonal antibody, ofatumumab. This work demonstrates that VCD is useful technique for monitoring subtle secondary structure changes of protein therapeutics that may occur during processing or handling. © 2013 Bentham Science Publishers.


PubMed | Legacy BioDesign LLC
Type: Journal Article | Journal: Journal of pharmaceutical sciences | Year: 2011

There are many aspects of stabilization of lyophilized proteins. Of these various factors, retention of native structure, having sufficient amount of stabilizer to embed the protein within an amorphous matrix, and dampening -relaxations have been shown to be critical in optimizing protein stability during storage. In this study, an IgG1 was lyophilized with varying amounts of sucrose. In some formulations, a small amount of sorbitol was added as a plasticizer. The structure of the protein in dried state was monitored using infrared (IR) spectroscopy. The IR spectra indicated increasing retention of the native structure, which correlated with stability as indicated by size-exclusion chromatography as well as micro-flow imaging. Maximal stability was achieved with a 2:1 mass ratio of sucrose to protein, which is more than that would be expected based on earlier studies. Analysis of both high and low frequency bands associated with intramolecular -sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the -sheet bands and the enthalpy of relaxation, suggesting that amide I bands can provide some indication of the degree of coupling to the sugar matrix, as well as structural heterogeneity of the protein.


PubMed | Legacy BioDesign LLC
Type: Journal Article | Journal: Pharmaceutical development and technology | Year: 2011

Covalent attachment of poly(ethylene) glycol (PEG) groups to proteins, a process commonly called PEGylation, is often used to improve the performance of a protein in vivo. To date, at least eight such PEGylated peptide and protein conjugates have been approved as therapeutic agents and many more have undergone clinical trials. This review examines PEGylation from the perspective of developing a commercially viable drug product. The first section focuses on obtaining a pure and well-characterized drug substance. The latter section discusses formulation and manufacturing issues, with an emphasis on analytical methodology that provides the most detailed description of the purity and stability of PEGylated proteins.

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