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.'

Application of Formulation Principles to Stability Issues Encountered During Processing, Manufacturing, and Storage of Drug Substance and Drug Product Protein Therapeutics.

The field of formulation and stabilization of protein therapeutics has become rather extensive. However, most of the focus has been on stabilization of the final drug product. Yet, proteins experience stress and degradation through the manufacturing process, starting with fermentaition. This review describes how formulation principles can be applied to stabilize biopharmaceutical proteins during bioprocessing and manufacturing, considering each unit operation involved in prepration of the drug substance. In addition, the impact of the container on stabilty is discussed as well.

Characterization of Oligomer Formation of Surfactant Protein-D (SP-D) Using AF4-MALLS.

BACKGROUND: Surfactant protein-S (SP-D) is a naturally occurring lung protein with the potential to treat pulmonary infections. A recombinant surfactant protein-D (SP-D) has been produced and was previously found to exist in multiple oligomeric states. INTRODUCTION: Separation and characterization of interconverting oligomeric states of a protein can be difficult using chromatographic methods, so an alternative separation technique was employed for SPD to characterize the different association states that exist. METHODS: Samples of SP-D were analyzed using asymmetrical flow field-flow fractionation (AF4) using UV and multi-angle laser light scattering (MALLS) detection. The AF4 method appears to be able to separate species as small as the monomer up to the dodecamer (the dominant species) to much larger species with a molar mass greater than 5 MDa. RESULTS: Consistent elution of four distinct peaks was observed after repeated injections. The largest species observed under the last peak (labeled as Peak 4) were termed "unstructured multimers" and were resolved fairly well from the other species. The AF4-MALLS data suggest that only a small fraction of Peak 4 truly corresponds to high molar mass unstructured multimers. All other peaks demonstrated significant molar mass homogeneity consistent with AFM results. CONCLUSION: AF4-MALLS technology appears to be a powerful analytical approach to characterize the complex and dynamic interplay among different protein oligomeric species of SP-D in an aqueous solution.

Chemometric Study of the Relative Aggregation Propensity of Position 19 Mutants of Aß(1-42).

BACKGROUND: The importance of aromaticity vs. hydrophobicity of the central hydrophobic core (CHC, residues 17-20) in governing fibril formation in Aß(1-42) has been the focus of an ongoing debate in the literature. INTRODUCTION: Mutations in the CHC (especially at Phe19 and Phe20) have been used to examine the relative impact of hydrophobicity and aromaticity on the degree of aggregation of Aß(1-42). However, the results have not been conclusive. METHODS: Partial least squares (PLS) modeling of aggregation rates, using reduced properties of a series of position 19 mutants, was employed to identify the physicochemical properties that had the greatest impact on the extent of aggregation. RESULTS: The PLS models indicate that hydrophobicity at position 19 of Aß(1-42) appears to be the primary and dominant factor in controlling Aß(1-42) aggregation, with aromaticity having little effect. CONCLUSION: This study illustrates the value of using reduced properties of amino acids in conjunction with PLS modeling to investigate mutational effects in peptides and proteins, as the reduced properties can capture in a quantitative manner the different physicochemical properties of the amino acid side chains. In this particular study, hydrophobicity at position 19 was determined to be the dominant property controlling aggregation, while size, charge, and aromaticity had little impact.

Analysis of Peptides using Asymmetrical Flow Field-flow Fractionation (AF4).

While asymmetrical flow field-flow fractionation (AF4) has been widely used for separation of high molecular weight species and even particles, its ability to resolve lower molecular weight species has rarely been explored. Over the course of many projects, we have discovered that AF4 can be an effective analytical method for separating peptides from oligomers and higher molecular weight aggregates. The methodology can be used even for peptides as small as 2 kD in molecular weight. Using multi-angle laser light scattering (MALLS) detection, accurate masses of the parent peptide can be obtained, provided accurate extinction coefficients are provided. It was shown that AF4 can be stability-indicating, suggesting that AF4-MALLS may be a suitable alternative to the use of SEC to monitor the aggregation of peptides.

A Chemometric Approach Toward Predicting the Relative Aggregation Propensity: Aß(1-42).

A number of algorithms have been developed to predict the aggregation propensity of peptides and proteins, but virtually none have the ability to provide sequence-specific information on what physicochemical properties are most important in altering aggregation propensity. In this study, a chemometric approach using reduced amino acid properties is used to examine the aggregation behavior of a highly amyloidogenic peptide, Aß(1-42). Specific residues are identified as being critical to the aggregation process. At each of these positions, the important physicochemical properties are identified that would either accelerate or inhibit fibril formation.

Shape Characterization of Subvisible Particles Using Dynamic Imaging Analysis.

Protein aggregates and subvisible particles (SbvP), inherently present in all marketed protein drug products, have received increasing attention by health authorities. Dynamic imaging analysis was introduced to visualize SbvP and facilitate understanding of their origin. The educational United States Pharmacopeia chapter <1787> emphasizes that dynamic imaging analysis could be used for morphology measurements in the size range of 4-100 µm. However, adequate morphology characterization, as suggested in the United States Pharmacopeia <1787> proposed size range, remains challenging as nonspherical size standards are not commercially available. In this study, a homogenous and well-defined nonspherical particle standard was fabricated and used to investigate the capabilities of 2 dynamic imaging analysis systems (microflow imaging (MFI) and FlowCAM) to characterize SbvP shape in the size range of 2-10 µm. The actual aspect ratio of the SbvP was measured by scanning electron microscopy and compared to the results obtained by dynamic imaging analysis. The test procedure was used to assess the accuracy in determining the shape characteristics of the nonspherical particles. In general, dynamic imaging analysis showed decreasing accuracy in morphology characterization for 5 µm and 2 µm particles. The test procedure was also capable to compare and evaluate differences between the 2 dynamic imaging methods. The present study should help to define ranges of operation for dynamic imaging analysis systems.

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.

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.

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.

Flow imaging: moving toward best practices for subvisible particle quantitation in protein products.

The potential impact of subvisible particles (SVPs) in protein therapeutic products has received a great deal of attention recently. As a result, new analytical methods have emerged to characterize and quantify SVPs. Among these, flow imaging (also called flow microscopy) has been widely employed. As we have used both FlowCAM® and Micro-Flow Imaging™ in a variety of development projects, we wished to share our experiences and observations, with the intention of fostering a discussion that will lead to best practices for the use of flow imaging to quantify SVP levels in biopharmaceuticals.

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.

Effects of solution conditions on methionine oxidation in albinterferon alfa-2b and the role of oxidation in its conformation and aggregation.

Physical and chemical degradation of therapeutic proteins can occur simultaneously. In this study, our first objective was to investigate how solution conditions that impact conformational stability of albinterferon alfa-2b, a recombinant fusion protein, modulate rates of methionine (Met) oxidation. Another objective of this work was to determine whether oxidation affects conformation and rate of aggregation of the protein. The protein was subjected to oxidation in solutions of varying pH, ionic strength, and excipients by the addition of 0.02% tertiary-butyl hydroperoxide (TBHP). The rate of formation of Met-sulfoxide species was monitored by reversed-phase high-performance liquid chromatography and compared across solution conditions. Albinterferon alfa-2b exhibited susceptibility to Met oxidation during exposure to TBHP that was highly dependent on solution parameters, but there was not a clear correlation between oxidation rate and protein conformational stability. Met oxidation resulted in significant perturbation of both secondary and tertiary structure of albinterferon alfa-2b as shown by both far-ultraviolet (UV) and near-UV circular dichroism. Moreover, oxidation of the protein caused a noticeable reduction in the protein's resistance to thermal denaturation. Surprisingly, despite its negative effect on solution structure and conformational stability, oxidation actually reduced the protein's aggregation rate during agitation at room temperature as well as during quiescent incubation at 40°C. Oxidation of the protein resulted in improved colloidal stability of the protein, which is manifested by a more positive B(22) value in the oxidized protein. Thus, the reduced aggregation rate after oxidation suggests that increased colloidal stability of oxidized albinterferon alfa-2b counteracted oxidation-induced decreases in conformational stability.

Physical stability of albinterferon-α(2b) in aqueous solution: effects of conformational stability and colloidal stability on aggregation.

Controlling aggregation in protein therapeutics is a significant challenge. In this study, the aggregation behavior of albinterferon-α(2b) , a genetic fusion protein combining human serum albumin and α-interferon, was examined as a function of solution conditions. The stability was monitored during agitation and during storage at elevated temperature, where the extent of aggregation was determined using size-exclusion chromatography. The osmotic second virial coefficient and the free energy of unfolding were measured for each sample. This study demonstrates that both increasing conformational stability and maximizing colloidal stability help to maintain the physical stability of albinterferon-α(2b).

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.

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.

Aggregation of recombinant human botulinum protein antigen serotype C in varying solution conditions: implications of conformational stability for aggregation kinetics.

Solution conditions greatly affect the aggregation rate of a protein. Elucidating these influences provides insight into the critical factors governing aggregation. In this study, recombinant human botulinum protein antigen serotype C [rBoNTC (H(c))] was employed as a model protein. rBoNTC (H(c)) aggregated irreversibly during incubation at 42°C. The aggregation rate was studied as a function of solution conditions, including varying the pH from 3.5 to 8.0 and with or without 150 mM NaCl, 7.5% (w/v) trehalose, and 0.5 M urea. Some solution conditions retarded rBoNTC (H(c)) aggregation, whereas others accelerated aggregation, particularly acidic pH and addition of NaCl or urea. To better understand the mechanism by which these solution conditions influenced aggregation rates, the structure of rBoNTC (H(c)) was characterized using circular dichroism, fluorescence, and ultraviolet absorbance spectroscopies. Conformational stability was assessed from equilibrium urea-induced unfolding studies and by using differential scanning calorimetry (DSC). The activation energy of the aggregation reaction (E(a)) was estimated from an analysis of the heating-rate dependence of the thermal transition observed during DSC heating scans. Overall, for rBoNTC (H(c)), an inverse correlation was found between conformational stability and aggregation rate, as well as between the kinetic barrier to unfolding (i.e., E(a)) and aggregation rate.