Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation.

PURPOSE: To determine the effect of moisture and the role of the glass transition temperature (Tg) on the stability of a high concentration, lyophilized, monoclonal antibody. METHODS: A humanized monoclonal antibody was lyophilized in a sucrose/histidine/polysorbate 20 formulation. Residual moistures were from 1 to 8%. Tg values were measured by modulated DSC. Vials were stored at temperatures from 5 to 50 degrees C for 6 or 12 months. Aggregation was monitored by size exclusion chromatography and Asp isomerization by hydrophobic interaction chromatography. Changes in secondary structure were monitored by Fourier transform infrared (FTIR). RESULTS: T. values varied from 80 degrees C at 1% moisture to 25 degrees C at 8% moisture, there was no cake collapse and were no differences in the secondary structure by FTIR. All formulations were stable at 5 degrees C. High moisture cakes had higher aggregation rates than drier samples if stored above their Tg values. Intermediate moisture vials were more stable to aggregation than dry vials. High moisture samples had increased rates of Asp isomerization at elevated temperatures both above and below their Tg values. Chemical and physical degradation pathways followed Arrhenius kinetics during storage in the glassy state. Only Asp isomerization followed the Arrhenius model above the Tg value. Both chemical and physical stability at T > or = Tg were fitted to Williams-Landel-Ferry (WLF) kinetics. The WLF constants were dependent on the nature of the degradation system and were not characteristic of the solid system. CONCLUSION: High moisture levels decreased chemical stability of the formulation regardless of whether the protein was in a glassy or rubbery state. In contrast, physical stability was not compromised, and may even be enhanced, by increasing residual moisture if storage is below the Tg value.

Identification of multiple sources of charge heterogeneity in a recombinant antibody.

Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.

Evidence for the involvement of histidine A(12) in the aggregation and precipitation of human relaxin induced by metal-catalyzed oxidation.

The metal-catalyzed oxidation (ascorbate/cupric chloride/oxygen) of recombinant human relaxin (rhRlx, type II) was shown by Li et al. [Li, S., Nguyen, T. H., Schöneich, C., and Borchardt, R. T. (1995) Biochemistry 34, 5762-5772] to result in the chemical modification of His A(12), Met B(4), and Met B(25). Considering the fact that His A(12) exists in an extended loop that joins two alpha-helices in this protein, we hypothesized that oxidation of this specific amino acid leads to alterations in the secondary and tertiary structures of the protein, resulting in the pH-dependent aggregation/precipitation phenomena observed in our earlier studies (i.e., at pH >6.0 most of the degradants of rhRlx are insoluble). Evidence obtained in the current study that supports this hypothesis includes the following: (i) oxidation of rhRlx with hydrogen peroxide (H(2)O(2)), which leads only to modification of Met B(4) and Met B(25), does not result in the pH-dependent aggregation/precipitation of the protein; and (ii) metal-catalyzed oxidation of porcine relaxin (pRlx), which does not contain His at position A(12), leads to chemical degradation of the protein [e.g., Met A(2) is oxidized] but produces only slight pH-dependent aggregation/precipitation of the protein. In addition, experimental evidence is provided to show that the physical instability of rhRlx observed at pH >6.0 does not appear to be related to the pH-dependent solubility of a common protein degradant. Instead, it appears that several oxidation products of His A(12) are produced in a pH-dependent manner and that these oxidation products produce different effects on the physical stability of the protein. Evidence in support of this conclusion includes the observation that the soluble degradants of rhRlx showed reduced levels of His, reduced levels of the T(2)-T(7) tryptic fragment that contained His A(12), and the presence of 2-oxo-His. Similarly, the precipitated degradants of rhRlx showed reduced levels of His but no 2-oxo-His. In addition, the soluble degradants, which contain 2-oxo-His, appear to exist as monomers having an average molecular weight similar to that of rhRlx. These results suggest that the metal-catalyzed oxidation of His A(12) leads to other, as yet unidentified oxidation products of His A(12) that affect the secondary/tertiary structure of the protein more significantly than does 2-oxo-His and ultimately lead to the physical instability of the protein observed at higher pH values.

Investigation of protein-surfactant interactions by analytical ultracentrifugation and electron paramagnetic resonance: the use of recombinant human tissue factor as an example.

PURPOSE: The purpose of this work is to utilize electron paramagnetic resonance (EPR) spectroscopy in conjunction with analytical ultracentrifugation (AUC) to investigate the binding of surfactants to proteins with a transmembrance domain. As an example these methods have been used to study the interaction of a nonionic surfactant, C12E8, to recombinant human tissue factor (rhTF) in liquid formulations. The complementary nature of the two techniques aids in data interpretation when there is ambiguity using a single technique. In addition to binding stoichiometries, the possibility of identifying the interacting domains by using two forms of rhTF is explored. METHODS: Two recombinant, truncated forms of human tissue factor were formulated in the absence of phospholipids. Neither of the recombinant proteins, produced in E. coli, contains the cytoplasmic domain. Recombinant human tissue factor 243 (rhTF 243) consists of 243 amino acids and includes the transmembrane sequences. Recombinant human tissue factor 220 (rhTF 220), however, contains only the first 221 amino acids of the human tissue factor, lacking those of the transmembrane region. EPR and AUC were used to investigate the interactions between these two forms of rhTF and polyoxyethylene 8 lauryl ether, C12E8. RESULTS: Binding of C12E8 to rhTF 243 is detected by both EPR spectroscopy and AUC. Although a unique binding stoichiometry was not determined, EPR spectroscopy greatly narrowed the range of possible solutions suggested by the AUC data. Neither technique revealed an interaction between rhTF 220 and C12E8. CONCLUSIONS: The complementary nature of EPR spectroscopy and AUC make the combination of the two techniques useful in data interpretation when studying the interactions between rhTF and C12E8. By utilizing these techniques in this study, the binding stoichiometry of rhTF 243 to C12E8 ranges from 1.2:1 to 1.3:0.6 based on an aggregation number of 120. This binding is consistent with previously reported activity data that showed an increase in clotting rate when rhTF 243 is in the presence of C12E8 micelles. From the rhTF 220 data, it can further be concluded that the transmembrane domain of rhTF is necessary for interactions with C12E8.

The effect of formulation excipients on protein stability and aerosol performance of spray-dried powders of a recombinant humanized anti-IgE monoclonal antibody.

PURPOSE: To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. METHODS: Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. RESULTS: Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300: 1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. CONCLUSIONS: Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.

Protein inhalation powders: spray drying vs spray freeze drying.

PURPOSE: To develop a new technique, spray freeze drying, for preparing protein aerosol powders. Also, to compare the spray freeze-dried powders with spray-dried powders in terms of physical properties and aerosol performance. METHODS: Protein powders were characterized using particle size analysis, thermogravimetric analysis, scanning electron microscopy, X-ray powder diffractometry, and specific surface area measurement. Aerosol performance of the powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger or an Anderson cascade impactor. Two recombinant therapeutic proteins currently used for treating respiratory tract-related diseases, deoxyribonuclase (rhDNase) and anti-IgE monoclonal antibody (anti-IgE MAb), were employed and formulated with different carbohydrate excipients. RESULTS: Through the same atomization but the different drying process, spray drying (SD) produced small (approximately 3 microns), dense particles, but SFD resulted in large (approximately 8-10 microns), porous particles. The fine particle fraction (FPF) of the spray freeze-dried powder was significantly better than that of the spray-dried powder, attributed to better aerodynamic properties. Powders collected from different stages of the cascade impactor were characterized, which confirmed the concept of aerodynamic particle size. Protein formulation played a major role in affecting the powder's aerosol performance, especially for the carbohydrate excipient of a high crystallization tendency. CONCLUSIONS: Spray freeze drying, as opposed to spray drying, produced protein particles with light and porous characteristics, which offered powders with superior aerosol performance due to favorable aerodynamic properties.

Influence of calcium ions on the structure and stability of recombinant human deoxyribonuclease I in the aqueous and lyophilized states.

The effect of calcium ions on the structure and stability of recombinant human DNase I (rhDNase) in the aqueous and solid (lyophilized) states was investigated. Fourier transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure, while chemical stability was monitored in terms of deamidation and soluble aggregate formation at 40 degrees C. The exogenous calcium was removed by EGTA. This process can remove all but approximately one calcium ion per protein molecule. Analysis of the FTIR spectra in the amide III region in either the aqueous or lyophilized state demonstrated that removal of exogenous Ca2+ by EGTA-treatment had little effect on the secondary structure (and lyophilization-induced rearrangement thereof). For the aqueous solution, circular dichroism was used as an independent technique and confirmed that there was no large overall change in the secondary or tertiary structure upon the removal of calcium. The primary degradation route for the aqueous protein was deamidation. For the EGTA-treated protein, there was also severe covalent aggregation, e.g., formation of intermolecular disulfides facilitated by the cleavage of Cys173-Cys209. The aggregates exhibited a markedly different secondary structure compared to the native protein. For instance, the beta-sheet band observed at ca. 1620 cm-1 wavenumber in the amide I second derivative spectra was increased. Enzymatic activity was completely lost upon aggregation, consistent with the cleavage of the aforementioned native disulfide. For the protein lyophilized in the presence of Ca2+, there was no increase in deamidated species during solid-state storage; however, some aggregation was observed. For the lyophilized EGTA-treated protein, aggregation was even more pronounced, and there was some loss in enzymatic activity upon reconstitution. Thus, the removal of calcium ions by EGTA-treatment decreased the stability of rhDNase in both the aqueous and solid states even though no large overall calcium-induced structural changes could be observed by the techniques used in this study.

Effect of mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody.

We have examined the stability and aerosol performance of the pharmaceutical protein recombinant humanized anti-IgE monoclonal antibody (rhuMAbE25) spray dried with mannitol. The aerosol performance was measured by the fine particle fraction (FPF), and stability was assessed by the formation of soluble aggregates. When mannitol was added to the spray-dried rhuMAbE25 formulation, its ability to stabilize the protein leveled off above about 20% (w/w, dry basis). The FPF of the spray-dried formulations was stable during storage for rhuMAbE25 containing 10% and 20% mannitol, but the 30% formulation exhibited a dramatic decrease upon storage at both 5 degreesC and 30 degreesC, due to mannitol crystallization. We tested the addition of sodium phosphate to a 60:40 rhuMAbE25:mannitol (w:w) mixture, which otherwise crystallized upon spray drying and yielded a nonrespirable powder. The presence of sodium phosphate was successful in inhibiting mannitol crystallization upon spray drying and dramatically lowering the rate of solid-state aggregation. However, over long-term storage some crystallization was observed even for the phosphate-containing samples, concomitantly with increased particle size and decreased suitability for aerosol delivery. Therefore, the physical state of mannitol (i.e., amorphous or crystalline) plays a role both in maintaining protein stability and providing suitable aerosol performance when used as an excipient for spray-dried powders. Agents which retard mannitol crystallization, e.g., sodium phosphate, may be useful in extending the utility of mannitol as an excipient in spray-dried protein formulations.

Effect of spray drying and subsequent processing conditions on residual moisture content and physical/biochemical stability of protein inhalation powders.

PURPOSE: To understand the effect of spray drying and powder processing environments on the residual moisture content and aerosol performance of inhalation protein powders. Also, the long-term effect of storage conditions on the powder's physical and biochemical stability was presented. METHODS: Excipient-free as well as mannitol-formulated powders of a humanized monoclonal antibody (anti-IgE) and recombinant human deoxyribonuclease (rhDNase) were prepared using a Buchi 190 model spray dryer. Residual moisture content and moisture uptake behavior of the powder were measured using thermal gravimetric analysis and gravimetric moisture sorption isotherm, respectively. Protein aggregation, the primary degradation product observed upon storage, was determined by size-exclusion HPLC. Aerosol performance of the dry powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger (MSLI). RESULTS: Spray-dried powders with a moisture level (approximately 3%) equivalent to the freeze-dried materials could only be achieved using high-temperature spray-drying conditions, which were not favorable to large-male manufacturing, or subsequent vacuum drying. These dry powders would equilibrate with the subsequent processing and storage environments regardless of the manufacturing condition. As long as the relative humidity of air during processing and storage was lower than 50%, powders maintained their aerosol performance (fine particle fraction). However, powders stored under drier conditions exhibited better long-term protein biochemical stability. CONCLUSIONS: Manufacturing, powder processing, and storage environments affected powder's residual moisture level in a reversible fashion. Therefore, the storage condition determined powder's overall stability, but residual moisture had a greater impact on protein chemical stability than on powder physical stability.

Interaction of human IgE with soluble forms of IgE high affinity receptors.

PURPOSE: Interaction of human IgE with its high affinity receptor (Fc epsilon RI) on mast cells and basophils is an important step for initiating IgE mediated immune responses. To characterize the IgE and Fc epsilon RI interaction, we investigated this interaction in terms of stoichiometry and binding affinity in solution. The binding of IgE and IgE Fc epsilon RI alpha chain, the extracellular portion of IgE high affinity receptor (sFc epsilon RI alpha) was compared with the binding of IgE and IgE immunoadhesin (Fc epsilon RI alpha-IgG). METHODS: The interaction was characterized by analytical ultracentrifugation, size exclusion chromatography, light scattering and ELISA. RESULTS: We show that the sFc epsilon RI alpha is only able to bind to one IgE, while the immunoadhesin can bind to two IgE. The interaction between IgE and Fc epsilon RI is very strong. Both forms of soluble receptors have similar intrinsic binding affinity with IgE. CONCLUSIONS: Both soluble receptors (Fc epsilon RI alpha-IgG and sFc epsilon RI alpha) can block the binding of IgE to its high affinity receptors on cell surface. The Fc epsilon RI alpha-IgG is a better IgE binding protein than sFc epsilon RI alpha at physiological relevant conditions. A humanized anti-IgE monoclonal antibody, rhuMAb E25 that also can block the binding of IgE to its high affinity receptors appears to bind to IgE at slightly different regions or in a different manner as the soluble forms of IgE receptors.

Characterization of complex formation by humanized anti-IgE monoclonal antibody and monoclonal human IgE.

The interaction of human IgE with high-affinity IgE Fc receptors on cells of the immune system plays an essential role in the type I hypersensitivity reaction. A proposed therapy is to use an anti-IgE monoclonal antibody to block the binding of IgE to its high-affinity receptor on mast cells and basophils, thus preventing subsequent release of the inflammatory agents after exposure to allergen. We report here the solution characteristics of immune complexes formed by a humanized anti-IgE monoclonal antibody (rhuMAb E25) and IgE using sedimentation analysis and size exclusion chromatography. We demonstrate that the rhuMAb E25 is able to form a variety of complexes with IgE at different molar ratios. The largest complex was identified by sedimentation equilibrium analysis as a heterohexamer with very high stability. The intermediate complex formed when one of the interacting components is in large molar excess appears to have a trimeric structure. The high-affinity interaction of rhuMAb E25 and IgE has also been confirmed. Furthermore, by using hydrodynamic modeling, we show that the largest complex may be represented by a cyclic structure.

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to recognize G/UAG repeats in the trpEDCFBA and trpG transcripts.

A filter binding assay was developed to study interactions between purified TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, and trp specific transcripts. TRAP formed stable complexes with trpEDCFBA leader RNA; binding was L-tryptophan-dependent and was complete within 60 s. TRAP binds to a segment of the trp leader transcript that includes part of an RNA antiterminator structure. Binding to this segment allows formation of an RNA terminator structure, thereby promoting transcription termination. Using several trpEDCFBA leader deletion transcripts, we identified several closely spaced trinucleotide repeats (seven GAG and four UAG repeats) in the trp leader transcript that appeared to be required for TRAP binding. We also showed that TRAP binds to a segment of the trpG transcript that includes the trpG ribosome binding site; the nucleotide sequence of this segment contains several appropriately spaced trinucleotide repeats (seven GAG, one UAG, and one AAG). TRAP binding to the trpG transcript would block translation initiation. RNA footprint analysis confirmed interaction between TRAP and the trinucleotide repeats in the various transcripts. TRAP, in the presence or absence of L-tryptophan, appears to consist of 11 or 12 identical 8-kDa subunits. Our findings suggest that each tryptophan-activated TRAP subunit can bind one G/UAG repeat in a target transcript. Multiple protein-RNA interactions are required for stable association.

Characterization of aerosols of human recombinant deoxyribonuclease I (rhDNase) generated by jet nebulizers.

Recombinant human deoxyribonuclease I (rhDNase) is a new therapeutic agent developed to improve clearance of purulent sputum from the human airways. It is delivered by inhalation. Four jet nebulizers, T Up-Draft II (Hudson), Customized Respirgard II (Marquest), Acorn II (Marquest), and Airlife Misty (Baxter), were evaluated in vitro for their ability to deliver aerosols of rhDNase. The aerosols were generated from 2.5-mL aqueous solutions of rhDNase, at concentrations of either 1 or 4 mg/mL. In all experiments, the Pulmo-Aide Compressor (De Vilbiss) was used to supply the air to the nebulizers. Between 20 and 28% of the rhDNase dose initially placed in the nebulizers was delivered to the mouthpiece in the respirable range (1-6 microns). Evaluation of the rhDNase following nebulization in all four devices indicated that there was no loss in enzymatic activity and no increase in aggregation. Circular dichroism spectrophotometry indicated there was no change in either the secondary or the tertiary structure in rhDNase following nebulization. These results show that all four nebulizers are essentially equivalent in their ability to deliver respirable doses of rhDNase in an intact, fully active form. Changing the concentration of the solution in the nebulizer from 4 to 1 mg/mL rhDNase leads to a proportional reduction in the respirable dose delivered to the mouthpiece.

Electron-microscopic and hydrodynamic characterization of recombinant apolipoprotein (a) and its association with LDL.

A recombinant apo(a) containing 17 kringle 4 domains as well as the kringle 5 and protease domains of apo(a) was characterized by hydrodynamic studies and electron microscopy. Recombinant apo(a) is a monomer in solution with a molecular weight of 325,000 by sedimentation equilibrium and 320,000 by sedimentation and diffusion, and it is a highly asymmetric molecule with a frictional ratio of 2.2. In the electron microscope recombinant apo(a) is visualized as a flexible chain of domains approximately 800 A long. Sedimentation velocity studies also demonstrate that when it is mixed with LDL, recombinant apo(a) reversibly forms an Lp(a)-like complex with a 1:1 stoichiometry; moreover, complex formation is inhibited by 6-amino hexanoic acid. Hydrodynamic modeling and electron microscopy suggest that only a small portion of the r-apo(a) molecule interacts with the LDL and the rest of the chain extends into solution. Preliminary studies indicate that recombinant apo(a) also binds mouse LDL.

Physical properties of recombinant apolipoprotein(a) and its association with LDL to form an LP(a)-like complex.

Recombinant apolipoprotein(a) has been studied by hydrodynamic techniques and electron microscopy. Recombinant apo(a) was primarily a monomer in solution with an s0(20,w) of 9.3 S, a D20,w of 2.29 ficks, and a molecular weight of 325,000 from sedimentation equilibrium and 318,000 from combining the sedimentation and diffusion coefficients. A small amount, approximately 10%, of the recombinant apo(a) was present as a high molecular weight aggregate. The Stokes radius of the monomer, determined either from the diffusion coefficient or by combining the sedimentation equilibrium data with the sedimentation velocity data, was 94 A. The frictional ratio was 2.2, suggesting a highly asymmetric or random coil structure. In the electron microscope, recombinant apolipoprotein(a) was visualized as a long, highly flexible chain of domains forming large, open coiled structures on the EM grid with contour lengths of about 800 A. Addition of 6-aminohexanoic acid at 50 mM, a concentration which should saturate the weak lysine binding sites, did not alter the sedimentation behavior. In vivo, apolipoprotein(a) is associated tightly with LDL to form a highly atherogenic lipoprotein, Lp(a). A single molecule of recombinant apo(a) also associated tightly with LDL to yield a 13.3-S Lp(a)-like complex. This complex dissociated upon the addition of 50 mM 6-aminohexanoic acid. A novel sucrose gradient centrifugation technique was employed to determine a dissociation constant for the reversible equilibrium between recombinant apo(a) and LDL; at physiological ionic strength the dissociation constant was 0.3 nM. Raising the salt concentration to 5 M NaBr caused the dissociation constant to increase to 500 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of the extracellular domain of the 75-kilodalton tumor necrosis factor receptor.

An expression plasmid encoding the extracellular domain of the 75-kDa human tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable cell line secreting soluble TNF-R2 (sTNF-R2). Purified sTNF-R2 was resolved by SDS-PAGE into one band of approximate M(r) 43,000, consistent with a molecular weight of 36,000 +/- 4800 obtained by sedimentation equilibrium analysis. The apparent molecular weight observed by gel filtration chromatography was approximately 136,000. Glycosylation analysis revealed that Asn-149 is fully glycosylated, while Asn-171 is incompletely glycosylated (approximately 50%), and that a proline-, serine-, and threonine-rich region (residues 175-234) contains O-linked carbohydrate structures. Scatchard analysis of [125I]TNF-alpha and [125I]TNF-beta binding to sTNF-R2 gave dissociation constants (Kd) of 0.3 and 0.75 nM, respectively, comparable to those observed for intact cell-surface TNF-R2. The sTNF-R2 was found to block the cytotoxicity of both TNF-alpha and TNF-beta in a murine L-M cell assay. The sizes of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes determined by gel filtration chromatography were approximately 322 and 204 kDa, respectively. The stoichiometry of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes were examined by size-exclusion chromatography, sedimentation equilibrium, and cross-linking. The data from these studies suggest that at least two molecules of sTNF-R2 can bind to a single TNF-alpha or TNF-beta trimer.

The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation.

The biochemical literature has been surveyed to present an overview of the three most common protein degradation pathways: protein aggregation, deamidation, and oxidation. The mechanisms for each of these degradation routes are discussed with particular attention given to the effect of formulation conditions such as pH, ionic strength, temperature, and buffer composition. Strategies to reduce protein degradation are also discussed. These strategies are based on an understanding of the degradation mechanisms and the effect of changes in the storage conditions and formulation components on the degradation. The effects of each of the degradation routes on pharmaceutically relevant properties such as biological activity, metabolic half-life, and immunogenicity are summarized. Predicting a priori the alteration of pharmaceutical properties caused by the three degradation routes is difficult, and must be determined on a case-by-case basis for each protein. The difficulty in predicting the effect of degradation and analyzing the temperature dependence of reaction rates in proteins results in longer development times for protein formulations than for small molecule formulations. Although the use of accelerated stability to predict protein shelf life is difficult, conditions are discussed whereby the Arrhenius equation can be used to shorten formulation development time.