Stability of Protein Pharmaceuticals: Recent Advances.

There have been significant advances in the formulation and stabilization of proteins in the liquid state over the past years since our previous review. Our mechanistic understanding of protein-excipient interactions has increased, allowing one to develop formulations in a more rational fashion. The field has moved towards more complex and challenging formulations, such as high concentration formulations to allow for subcutaneous administration and co-formulation. While much of the published work has focused on mAbs, the principles appear to apply to any therapeutic protein, although mAbs clearly have some distinctive features. In this review, we first discuss chemical degradation reactions. This is followed by a section on physical instability issues. Then, more specific topics are addressed: instability induced by interactions with interfaces, predictive methods for physical stability and interplay between chemical and physical instability. The final parts are devoted to discussions how all the above impacts (co-)formulation strategies, in particular for high protein concentration solutions.'

Managing urban riverscapes: An assessment framework to integrate social-ecological values and physical processes.

The services that rivers provide and how they affect the landscape plays a dominate role in urban planning and development. Urban riverscapes, which consist of stream channels, their floodplains, biotic communities, and manmade features, are complex social-ecological and hydrogeomorphic systems. Yet, despite recognition of their place and value, rivers are often degraded in urban settings. Successfully managing urban riverscapes requires improved methods to assess them and to more effectively link stressors to values, and to incorporate these considerations in planning. Assessment of urban riverscapes' physical condition and function-a hydrogeomorphic assessment-is necessary to make these links, and inform more appropriate management strategies for sustainable and valued riverscape systems. The framework and methods used for such an assessment should be appropriate to the urban context, insofar as they are applicable to a range of streams from lightly degraded to highly utilized or constructed. Above all, the framework must prioritize the connection of human communities to riverscapes. In this article, we outline a framework for urban riverscape assessment which considers four facets of urban riverscapes: human values, hydrology, geomorphology, and ecology. The four facets, assessed across multiple nested scales, provide a flexible basis for context-driven hydrogeomorphic assessment, which is vital to informing better planning and management of urban riverscapes. The framework can be integrated with other facets (e.g. geochemical, aquatic ecology) depending on the scope of the assessment. By linking intrinsic, relational, and use-based values to physical conditions, watershed managers can select relevant and measurable indicators that directly inform interventions in the riverscape, catchment, or urban zones to improve riverscape function and urban vitality through planning mechanisms. This assessment framework facilitates dialogue between managers, practitioners, scientists, and the community; enabling technical and non-technical inputs to the development of assessment criteria, and a shared vision to inform targets and goals.

Detection of Transient Bursts in the EEG of Preterm Infants using Time-Frequency Distributions and Machine Learning.

Short-duration bursts of spontaneous activity are important markers of maturation in the electroencephalogram (EEG) of premature infants. This paper examines the application of a feature-less machine learning approach for detecting these bursts. EEGs were recorded over the first 3 days of life for infants with a gestational age below 30 weeks. Bursts were annotated on the EEG from 36 infants. In place of feature extraction, the time-series EEG is transformed into a time-frequency distribution (TFD). A gradient boosting machine is then trained directly on the whole TFD using a leave-one-out procedure. TFD kernel parameters, length of the Doppler and lag windows, are selected within a nested cross-validation procedure during training. Results indicate that detection performance is sensitive to Doppler-window length but not lag-window length. Median area under the receiver operator characteristic for detection is 0.881 (inter-quartile range 0.850 to 0.913). Examination of feature importance highlights a critical wideband region <15 Hz in the TFD. Burst detection methods form an important component in any fully-automated brain-health index for the vulnerable preterm infant.

High throughput detection of deamidation using S-(5'-adenosyl)-l-homocysteine hydrolase and a fluorogenic reagent.

Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate.

Denaturation and Aggregation of Interferon-τ in Aqueous Solution.

PURPOSE: To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride. METHODS: Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system. RESULTS: The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1. CONCLUSION: Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.

Role of Buffers in Protein Formulations.

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.

Stability of lyophilized teriparatide, PTH(1-34), after reconstitution.

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.

Comparability of higher order structure in proteins: chemometric analysis of second-derivative amide I Fourier transform infrared spectra.

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.

Use of the amide II infrared band of proteins for secondary structure determination and comparability of higher order structure.

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.

Stability of lyophilized sucrose formulations of an IgG1: subvisible particle formation.

Eight lyophilized formulations of a IgG1 monoclonal antibody (MAb) were prepared containing increasing levels of sucrose. In addition, three of the formulations had sorbitol added at a level of 5% w/w relative to sucrose. The samples were stored for up to 4 weeks at 40°C, which is well below the Tg. Upon reconstitution, the levels of subvisible particles were measured using microflow imaging (MFI). The formulation containing no sucrose contained exceedingly high levels of subvisible particles, accounting for as much as 25% of the weight of the protein. Addition of sucrose markedly decreased the number of subvisible particles, with the maximal sucrose:protein weight ratio being 2:1 (the highest level tested). Addition of sorbitol further decreased subvisible particle levels, even for formulations where the sucrose:protein ratio was relatively high. This suggests that even small amounts of a plasticizer like sorbitol can improve the storage stability of a lyophilized antibody formulation, probably by dampening ß-relaxations within the amorphous glass.

Characterization of protein higher order structure using vibrational circular dichroism spectroscopy.

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.

Comparability of protein therapeutics: quantitative comparison of second-derivative amide I infrared spectra.

Comparability determination for protein therapeutics requires an assessment of their higher order structure, usually by using spectroscopic methods. One of the most common techniques used to determine secondary structure composition of proteins is analysis of the second derivative of the amide I region of Fourier transform infrared (FTIR) spectra. A number of algorithms have been described for quantitative comparison of second-derivative amide I FTIR spectra, but no systematic evaluation has been conducted to assess these approaches. In this study, the two most common methods, spectral correlation coefficient and area of overlap (AO), are compared for their ability to determine spectral comparability of a protein as a function of changes in pH or temperature. Two other algorithms were considered as well. Recently, a QC compare similarity function found in OMNIC software has been reported as being useful in comparing amide I FTIR spectra. In addition, a new algorithm, termed modified AO, is described herein. These four methods were evaluated for their ability to determine comparability for second-derivative amide I FTIR spectra of four model proteins. The result is a framework for quantitative determination of whether any two spectra differ significantly.

Competitive, non-competitive, and mixed format cleavable tag immunoassays.

Immunoassays are one of the most useful diagnostic techniques in disease assessment, drug metabolite analysis, and environmental applications due largely in part to the selectivity and sensitivity provided by antibody-antigen interactions. Here, a multiplexed immunoassay termed cleavable tag immunoassay (CTI) was performed in competitive, non-competitive, and mixed formats for the analysis of proteins and small molecule biomarkers of inflammation and tissue damage. Microchip capillary electrophoresis (MCE) with fluorescence detection was employed for the analysis of fluorescently labeled tags corresponding to the analytes of interest cleaved from the detection antibodies. For this work we have selected 3-nitrotyrosine (3-NT) a molecule indicative of reactive nitrogen species (RNS), thyroxine (T4) a molecule used to monitor thyroid gland function, and C-reactive protein (CRP) a marker of chronic inflammation as model analytes to demonstrate the assay principles. The simultaneous detection of 3-nitrotyrosine (3-NT) and thyroxine (T4) was carried out as a proof-of-principle for the competitive CTI while non-competitive CTI performance was demonstrated via the analysis of C-reactive protein (CRP). Limit of detections (LOD) and dynamic ranges were investigated. LOD for 3-NT, T4, and CRP were 0.5µg/mL, 23nM, and 5µg/mL, respectively thus demonstrating the ability of the CTI to detect proteins and small molecules within clinical reference ranges. Moreover, this is the first report of the use of mixed format CTI chemistry for the simultaneous detection of proteins (CRP) and small molecules (3-NT) in a single assay. The success of this work demonstrates the ability of CTI to analyze intact proteins and small molecule biomarkers simultaneously.

Structure, stability, and mobility of a lyophilized IgG1 monoclonal antibody as determined using second-derivative infrared spectroscopy.

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 ß-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 ß-sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the ß-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.

Product development issues for PEGylated proteins.

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.

Stability of protein pharmaceuticals: an update.

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.

Analysis of oxidative stress biomarkers using a simultaneous competitive/non-competitive micromosaic immunoassay.

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 microL sample and 45 min for completion. Logistic curve fits and LOD (limits of detection) statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

Competitive immunoassays for simultaneous detection of metabolites and proteins using micromosaic patterning.

New high-throughput immunoassay methods for rapid point-of-care diagnostic applications represent an unmet need and current focus of numerous innovative methods. We report a new micromosaic competitive immunoassay developed for the analysis of the thyroid hormone thyroxine (T4), inflammation biomarker C-reactive protein (CRP), and the oxidative damage marker 3-nitrotyrosine (BSA-3NT) on a silicon nitride substrate. To demonstrate the versatility of the method, both direct and indirect format competitive immunoassays were developed and could be applied simultaneously for single samples. Signals from standard solutions were fit to a logistic equation, allowing simultaneous detection of T4 (7.7-257.2 nM), CRP (0.3-4.2 microg/mL), and BSA-3NT (0.03-22.3 microg/mL). Total assay time including sample introduction, washing, and fluorescence measurement was less than 45 min. Dissociation constants for affinity pairs in the system have been estimated using regression. This proof-of-concept experiment shows that both small and macromolecular biomarkers can be quantified from a single sample using the method and suggests that groups of clinically related analytes may be analyzed by competitive micromosaic immunoassay techniques.

Detection of cardiac biomarkers using micellar electrokinetic chromatography and a cleavable tag immunoassay.

Biomarkers provide clinicians with an important tool for disease assessment. Many different biomarkers have been discovered, but few of them suffice as stand-alone indicators for disease presence or prognosis. Because no single biomarker can be relied upon for accurate disease detection there has been a substantial push for new multianalyte screening methods. Furthermore, there is a need to push assays toward a point-of-care technology to reduce the time between clinical analysis and medical intervention and minimize artifacts created during sample storage. There currently are, however, few inexpensive multianalyte methods for disease detection that can function in a point-of-care setting. A new approach which bridges the gap between traditional immunoassays and high-density microarrays by utilizing microfluidics, immunoassays, and micellar electrokinetic chromatography (MEKC) is discussed here. This chemistry, the cleavable tag immunoassay (CTI), is a low- to medium-density heterogeneous immunoassay designed to detect 1-20 analytes simultaneously. Although similar to traditional sandwich immunoassays, this approach is unique because the signal is not imaged on the surface; instead, a fluorescent tag is chemically cleaved from the antibody and analyzed by microchip MEKC. In this report, the CTI chemistry is used for the detection of four cardiac biomarkers elevated in acute myocardial infarction. Limit of detection (LOD) and dynamic range are reported for all biomarkers with LODs on the order of low nanograms per milliliter to low picograms per milliliter. Most importantly, the dynamic range for each of the biomarkers spans the boundary between normal and elevated levels. Finally, elevated marker levels were measured in spiked human serum samples.