John F. Carpenter

Application of infrared spectroscopy to development of stable lyophilized protein formulations

The preparation of protein therapeutics as lyophilized (freeze-dried) products is often essential to obtain the requisite stability during shipping and long-term storage. When prepared properly, lyophilized proteins can retain stability for months or even years at ambient temperature [1]. A crucial aspect of proper, rational development of a lyophilized formulation is the recognition that the most sensitive element in the system is the protein itself. Unfortunately, in the early days of preparing lyophilized protein formulations, it appears that frequently the focus was primarily on obtaining an acceptable dried cake structure, with the apparent assumption that a protein would be resistant to freezing and dehydration stresses. For example, when only mannitol is employed as an excipient and crystalline bulking agent, the resulting cake structure is usually excellent, but proteins derive essentially no protection from crystalline mannitol [1]. Such approaches, which can be sufficient for more stable low molecular weight drugs, led to dried products in which the protein was so susceptible to physical and/ or chemical degradation that shipping and storage at subzero temperatures was required. Obviously, the potential advantages of a lyophilized product are mostly lost when there are such stringent requirements for control of product temperature. It is now well known that without adequate stabilization by the appropriate amorphous excipients (e.g. sucrose), the protein can be irreversibly denatured after lyophilization and reconstitution and/or after long-term storage in the dried solid and rehydration [1–8]. Since most protein drugs are delivered parenterally, even if only a small fraction of the total protein molecular population (e.g. a few percent) is irreversibly denatured and aggregated, then the product will not be acceptable. Until recently the changes in protein structure arising during the processing steps of lyophilization, which were usually manifested as protein aggregation after rehydration, were unknown. However, with infrared spectroscopy it is now possible to examine directly the secondary structure of a protein in the initial aqueous solution, and in both the frozen state and the final dried solid. This analysis, combined with exploitation of stabilizers that are specific for either freezing or drying stresses, has documented that both freezing and dehydration can induce protein unfolding [1–8]. Unfolding not only can lead to irreversible protein denaturation, if the sample is rehydrated immediately, but, perhaps more importantly for industrial development of lyophilized protein drugs, can also reduce storage stability in the dried solid [1,6,7]. Moreover, simply obtaining a native protein in samples rehydrated immediately after lyophilization is not necessaEuropean Journal of Pharmaceutics and Biopharmaceutics 45 (1998) 231–238

Understanding and Modulating Opalescence and Viscosity in a Monoclonal Antibody Formulation

Opalescence and high viscosities can pose challenges for high concentration formulation of antibodies. Both phenomena result from protein-protein intermolecular interactions that can be modulated with solution ionic strength. We studied a therapeutic monoclonal antibody (mAb) that exhibits high viscosity in solutions at low ionic strength ( approximately 20 cP at 90 mg/mL and 23 degrees C) and significant opalescence at isotonic ionic strength (approximately 100 nephelometric turbidity units at 90 mg/mL and 23 degrees C). The intermolecular interactions responsible for these effects were characterized using membrane osmometry, static light scattering, and zeta potential measurements. The net protein-protein interactions were repulsive at low ionic strength ( approximately 4 mM) and attractive at isotonic ionic strengths. The high viscosities are attributed to electroviscous forces at low ionic strength and the significant opalescence at isotonic ionic strength is correlated with attractive antibody interactions. Furthermore, there appears to be a connection to critical phenomena and it is suggested that the extent of opalescence is dependent on the proximity to the critical point. We demonstrate that by balancing the repulsive and attractive forces via intermediate ionic strengths and by increasing the mAb concentration above the apparent critical concentration both opalescence and viscosity can be simultaneously minimized.

Immunogenicity of aggregates of recombinant human growth hormone in mouse models.

Aggregation of recombinant therapeutic protein products is a concern due to their potential to induce immune responses. We examined the immunogenicity of protein aggregates in commercial formulations of recombinant human growth hormone produced by freeze-thawing or agitation, two stresses commonly encountered during manufacturing, shipping and handling of therapeutic protein products. In addition, we subjected each preparation to high-pressure treatment to reduce the size and concentration of aggregates present in the samples. Aggregates existing in a commercial formulation, as well as aggregates induced by freeze-thawing and agitation stresses enhanced immunogenicity in one or more mouse models. The use of high-pressure treatment to reduce size and concentrations of aggregates within recombinant human growth hormone formulations reduced their overall immunogenicity in agreement with the immunon hypothesis.

Cryopreservation of Stem Cells Using Trehalose: Evaluation of the Method Using a Human Hematopoietic Cell Line

While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.

Protein adsorption and excipient effects on kinetic stability of silicone oil emulsions

The effect of silicone oil on the stability of therapeutic protein formulations is of concern in the biopharmaceutical industry as more proteins are stored and delivered in prefilled syringes. Prefilled syringes provide convenience for medical professionals and patients, but prolonged exposure of proteins to silicone oil within prefilled syringes may be problematic. In this study, we characterize systems of silicone oil-in-aqueous buffer emulsions and model proteins in formulations containing surfactant, sodium chloride, or sucrose. For each of the formulations studied, silicone oil-induced loss of soluble protein, likely through protein adsorption onto the silicone oil droplet surface. Excipient addition affected both protein adsorption and emulsion stability. Addition of surfactant stabilized emulsions but decreased protein adsorption to silicone oil microdroplets. In contrast, addition of sodium chloride increased protein adsorption and decreased emulsion stability. Silicone oil droplets with adsorbed lysozyme rapidly agglomerated and creamed out of suspension. This decrease in the kinetic stability of the emulsion is ascribed to surface charge neutralization and a bridging flocculation phenomenon and illustrates the need to investigate not only the effects of silicone oil on protein stability, but also the effects of protein formulation variables on emulsion stability.

Aggregation of a Monoclonal Antibody Induced by Adsorption to Stainless Steel

Stainless steel is a ubiquitous surface in therapeutic protein production equipment and is also present as the needle in pre‐filled syringe biopharmaceutical products. Stainless steel microparticles can cause the aggregation of a monoclonal antibody (mAb). The initial rate of mAb aggregation was second order in steel surface area and zero order in mAb concentration, generally consistent with a bimolecular surface aggregation being the rate‐limiting step. Polysorbate 20 (PS20) suppressed the aggregation yet was unable to desorb the firmly bound first layer of protein that adsorbs to the stainless steel surface. Also, there was no exchange of mAb from the first adsorbed layer to the bulk phase, suggesting that the aggregation process actually occurs on subsequent adsorption layers. No oxidized Met residues were detected in the mass spectrum of a digest of a highly aggregated mAb, although there was a fourfold increase in carbonyl groups due to protein oxidation. Biotechnol. Bioeng. 2010;105: 121–129.

Meeting report on protein particles and immunogenicity of therapeutic proteins: Filling in the gaps in risk evaluation and mitigation

This meeting was successful in achieving its main goals: (1) summarize currently available information on the origin, detection, quantification and characterization of sub-visible particulates in protein products, available information on their clinical importance, and potential strategies for evaluating and mitigating risk to product quality, and (2) foster communication among academic, industry, and regulatory scientists to define the capabilities of current analytical methods, to promote the development of improved methods, and to stimulate investigations into the impact of large protein aggregates on immunogenicity. There was a general consensus that a considerable amount of interesting scientific information was presented and many stimulating conversations were begun. It is clear that this aspect of protein characterization is in its initial stages. As the development of these new methods progress, it is hoped that they will shed light on the role of protein particulates on product quality, safety, and efficacy. A topic which seemed appropriate for short term follow up was to hold further discussions concerning the development and preparation of one or more standard preparations of protein particulates. This would be generally useful to facilitate comparison of results among different studies, methods, and laboratories, and to foster further development of a common understanding among laboratories and health authorities which is essential to making further progress in this emerging field.

Adsorption of monoclonal antibodies to glass microparticles

Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension.

Role of electrostatic repulsion on colloidal stability of Bacillus halmapalus alpha-amylase

The colloidal stability of charged particles in suspension is often controlled by electrostatic repulsion, which can be rationalized in a semi-quantitative way by the DLVO theory. In the current study, we investigate this approach towards understanding irreversible protein aggregation, using Bacillus halmapalus alpha-amylase (BHA) as a model protein. Repulsive forces between partly unfolded monomers were shown to strongly affect aggregation. Adding salt, increasing valence of counter ions or decreasing pH in the direction of pI resulted in a shift in the rate-limiting step from association to unfolding as evidenced by a change in aggregation kinetics from second to first-order in protein concentration. Charge screening effects by salts resulted in increased average size of protein aggregates but only moderately affected the secondary structure of protein within the aggregates. Salt and pH effects could be explained within the DLVO framework, indicating that partially unfolded BHA monomers can be modelled realistically as colloids with a random charge distribution.

Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.

Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450±230 CFU-GM, 430±140 BFU-E, and 50±40 CFU-GEMM per 50 µL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25°C in the dark. Cells reconstituted immediately after lyophilization produced 580±90 CFU-GM (∼40%, relative to unprocessed controls p<0.0001), 170±70 BFU-E (∼40%, p<0.0001), and 41±22 CFU-GEMM (∼82%, pu200a=u200a0.4171), and cells reconstituted after 28 days at room temperature produced 513±170 CFU-GM (∼35%, relative to unprocessed controls, p<0.0001), 112±68 BFU-E (∼26%, p<0.0001), and 36±17 CFU-GEMM (∼82%, pu200a=u200a0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25°C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration.