Byeong S. Chang

Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony‐stimulating factor

We studied the non‐native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (ΔGunf) and osmotic second virial coefficient (B22), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ΔGunf and negative B22), the first step is rate‐limiting, and increasing ΔGunf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B22 values) the second step is rate‐limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate‐limiting.

Inhibition of stress-induced aggregation of protein therapeutics

Publisher Summary This chapter describes the types of stresses and conditions that are routinely found to cause aggregation of purified therapeutic proteins. Stresses encountered during processing include short-term exposure to high temperatures during pasteurization of aqueous solutions, freeze-thawing, freeze-drying, and exposure to denaturing interfaces because of agitation, filtration, air bubble entrainment during filling, and so on. It also considers the effects of long-term storage on protein stability in aqueous solutions and dried solids. Each section describes how the rational choice of stabilizing additives can be used to inhibit protein aggregation. These choices are based on a clear understanding of the mechanisms by which different additives succeed or fail as protein stabilizers under different conditions. The mechanisms are considered in detail and are illustrated in the chapter by selected examples. Finally, this chapter briefly describes a model that allows the calculation of the degree of expansion of the native state needed to form an aggregate-fostering species in aqueous solution and the utility of infrared spectroscopy to characterize the structure of proteins in precipitates.

Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native‐state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native‐state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far‐ and near‐UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native‐state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl‐induced unfolding, increased in proportion to sucrose concentration. Native‐state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange‐resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large‐scale than on small‐scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native‐state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.

Development of a Stable Freeze-dried Formulation of Recombinant Human Interleukin-1 Receptor Antagonist

AbstractPurpose. A formulation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was developed that provided both acute protection during lyophilization and storage stability in the dried solid. Methods. The formulation was optimized by monitoring the impact of excipients on protein degradation which was analyzed by turbidimetry and cation-exchange HPLC. Results. The most appropriate pH was 6.5. Sodium citrate buffer provided better stability than sodium phosphate buffer. Glycine was selected as a bulking agent because the greatest protein stability was noted when this bulking agent was used in combination with an amorphous protein stabilizer. Among the amorphous stabilizers tested, sucrose protected rhIL-lra best in the presence of glycine. When the protein was freeze-dried in the presence of an inadequate mass ratio of sucrose/protein (< 0.3), the rate of degradation of rhIL-lra increased. For a formulation containing 100 mg/ml of rhIL-lra, increasing the sucrose/protein mass ratio to ≥ 0.3 greatly increased storage stability. The moisture content of the dried solid affected the storage stability to a minor degree. Three different stoppers obtained from the WEST Company did not affect the stability of rhIL-lra. Conclusions. An optimized formulation could be reconstituted without precipitation after 14 months at 30 or 50°C. At 30°C, there was no loss of native protein due to deamidation, and only a 4% loss at 50°C. These results indicated that the optimized formulation could be stored at ambient temperatures for long periods, without damage to the protein.

Practical Approaches to Protein Formulation Development

As is the case with other pharmaceuticals, formulation development is one of the critical steps in developing a protein as a therapeutic product. Development of stable protein formulations may require even more resources and effort than conventional small molecule pharmaceuticals. Proteins typically have more stability issues as a result of their complexity and delicate structural stability. Fortunately, a great deal of research regarding protein stability has been conducted and this information is readily available in the literature (reviewed by Manning et al., 1989; Chen, 1992; Ahern and Manning, 1992a; Ahern and Manning, 1992b; Arakawa et al., 1993; Cleland et al., 1993; Wang and Pearlman, 1993; Pearlman and Wang, 1996; Volkin and Middaugh, 1997). Ultimately, it would be ideal to be able to develop a pure pharmaceutical containing only the native protein. However, it is not practical to have only the native form of a protein in the formulation because the protein must be purified from a complex biological mixture containing a pool of other proteins which includes misfolded, denatured, and degraded forms of the same protein. Furthermore, a major challenge is to maintain the integrity of the purified protein during routine pharmaceutical processing, storage, handling, and delivery to the patient. One could envision achieving this goal by developing a formulation with perfect stability, i.e., no physical and chemical change in the protein. Becauise proteins are complex molecules composed of numerous reactive chemical groups and delicate three-dimensional structures, identifying a set of conditions to keep all components stable is virtually impossible. In general, commercial therapeutic protein formulations are developed under the assumption that some degree of physicochemical changes will occur during storage and handling.

Development of an Efficient Single-Step Freeze-Drying Cycle for Protein Formulations

AbstractPurpose. An efficient freeze-drying cycle for recombinant human interleukin-1 receptor antagonist (rhIL-lra) formulations, which contained glycine and sucrose as excipients, was developed. Methods. Development was based on characterizing the frozen formulations by thermal analysis and by examining the effect of various lyophilization process parameters on the sublimation rate of ice. Results. Thermal analysis showed that the metastable glass of glycine in frozen formulation could be devitrified by slowly warming the frozen product to −15°C. During drying, the sublimation rate of ice was increased as a linear function of the difference between the vapor pressure of ice at the product temperature (PO) and the chamber pressure (PC). Therefore, the product temperature (Tp) was maintained as high as possible at temperatures below Tg′ of the formulation, in order to maximize the PO without allowing the collapse of cake. Although various combinations of shelf temperatures and chamber pressures could be used to obtain the same Tp, the combination of higher shelf temperature and lower chamber pressure was used to maximize sublimation rate. Conclusions. A single-step drying cycle was developed to take advantage of these observations. The shelf temperature was set for the secondary drying and the product temperature during primary drying was maintained below Tg′ by adjusting the chamber pressure. As the sublimation completed, the product temperature increased naturally to the shelf temperature for the secondary drying. This process resulted in successful drying of 1 ml of rhIL-lra formulation to 0.4% moisture content within 6 hours.

Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure

High hydrostatic pressure (HHP)‐mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three Gram‐negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox‐shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox‐shuffling agent resulted in solubilization yields of ∼42%–58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L‐arginine (0.5 M), Tween 20 (0.1 mM), or Triton X‐100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (∼70%–100%) to achieve refolding yields of ∼55%–78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.

Interactions between PEG and type I soluble tumor necrosis factor receptor: Modulation by pH and by PEGylation at the N terminus

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF‐RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF‐RI bound free PEG in a pH‐dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp‐107, at the C terminus, with the pH‐dependent and PEGylation‐dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF‐RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp‐107. Deprotonation of His‐105, which is perpendicular to Trp‐107, was integral to the binding mechanism producing a pH‐dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.

Stabilization of lyophilized porcine pancreatic elastase.

Porcine pancreatic elastase, a well-characterized serine protease, has been used as a model to assess the effects of excessive humidity on solid-state stability of the lyophilized protein. Elastase lyophilized without excipients retained full activity immediately after freeze-drying but became denatured upon continued storage at 40°C, 75% relative humidity. The extent of inactivation could be monitored through assays of amidolytic activity, as well as through changes in the circular dichroism (CD) and fluorescence spectra. Differential scanning calorimetry (DSC) was employed as a means of screening potential stabilizing additives; based on the results, sucrose and dextran 40 were selected for further evaluation. Both additives were effective in preventing denaturation. Possible mechanisms for the denaturation and stabilization of elastase are discussed.