Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event.

In spite of extensive research, protein aggregation still remains one of the most difficult phenomena to be understood in the field of biologics research and development. Protein aggregation is a complex process which results in the formation of a variety of supramolecular protein structures. Nucleation is the core step that initiates the cascade of molecular events leading to the formation of protein aggregates. Understanding and characterizing nucleation is therefore crucial to avoid undesired protein aggregation. Here we review the state of the art on protein aggregation in biotherapeutics, primarily focusing on the nucleation events, stimulating discussions about key open questions, and clarifying the peculiarities of aggregation process relative to other protein phase separation processes, such as crystallization. We summarize recent progress in the identification of the sources of protein aggregation and in the development of analytical tools to characterize this process. Moreover, we discuss significant gaps in the analysis and understanding of nucleation in non-native aggregation of biologics.

Stress Factors in Primary Packaging, Transportation and Handling of Protein Drug Products and Their Impact on Product Quality.

Protein-based biologic drugs encounter a variety of stress factors during drug substance (DS) and drug product (DP) manufacturing, and the subsequent steps that result in clinical administration by the end user. This article is the third in a series of commentaries on these stress factors and their effects on biotherapeutics. It focuses on assessing the potential negative impact from primary packaging, transportation, and handling on the quality of the DP. The risk factors include ingress of hazardous materials such as oxidizing residuals from the sterilization process, delamination- or rubber stopper-derived particles, silicone oil droplets, and leachables into the formulation, as well as surface interactions between the protein and packaging materials, all of which may cause protein degradation. The type of primary packaging container used (such as vials and prefilled syringes) may substantially influence the impact of transportation and handling stresses on DP Critical Quality Attributes (CQAs). Mitigations via process development and robustness studies as well as control strategies for DP CQAs are discussed, along with current industry best practices for scale-down and in-use stability studies. We conclude that more research is needed on postproduction transportation and handling practices and their implications for protein DP quality.

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality.

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.

Emerging Challenges and Innovations in Surfactant-mediated Stabilization of Biologic Formulations.

Biologics may be subjected to various destabilizing conditions during manufacturing, transportation, storage, and use. Therefore, biologics must be appropriately formulated to meet their desired quality target product profiles. In the formulations of protein-based biologics, one critical component is surfactant. Polysorbate 80 and Polysorbate 20 remain the most commonly used surfactants. Surfactants can stabilize proteins through different mechanisms and help the proteins withstand destabilization stresses. However, the challenges associated with surfactants, for instance, impurities, degradation, and potential triggering of adverse immune responses, have been encountered. Therefore, there are continued efforts to develop novel surfactants to overcome these challenges associated with traditional surfactants. Meanwhile, surfactants have also found their use in formulations of newer and novel modalities, namely, antibody-drug conjugates, bispecific antibodies, and adeno-associated viruses (AAV). This review provides an updated in-depth discussion of surfactants in the above-mentioned areas, namely mechanism of action of surfactants, a critical review of challenges with surfactants and current mitigation approaches, and emerging technologies to develop novel surfactants. In addition, gaps, current mitigations, and future directions have been presented to trigger further discussion and research to facilitate the use and development of novel surfactants.

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy.

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations.

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).

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.

Effect of buffer species on the thermally induced aggregation of interferon-tau.

It is now becoming apparent that a common pathway of protein aggregation involves the unimolecular structural rearrangement from the native state to a slightly expanded aggregation-competent species. It is the goal of this study to understand the aggregation and the effects of buffer on the stability of IFN-tau. In this study, the thermally-induced aggregation of interferon-tau (IFN-tau) is described. By monitoring the aggregation rate in the presence of increasing amounts of sucrose, the relative change in surface area (Deltas) for conversion to the aggregation-competent state can be determined. Under conditions of pH 7 and in 20 mM buffer, the protein displays different aggregation rates depending on the nature of the buffer species. The protein aggregates mostly quickly in phosphate buffer, slower in the presence of Tris and slowest in the presence of histidine. The largest value for Deltas occurs for the histidine-containing samples, where aggregation proceeds via a slightly expanded aggregation competent state with a surface area increase of 7.6%. Furthermore, it appears that histidine binds to the native state of IFN-tau, thereby stabilizing the native state and retarding aggregation. Measurement of the second virial coefficient, B(22), for different formulations indicates that inclusion of histidine has only a small effect on repulsion between protein molecules, suggesting that colloidal stabilization is not the dominant mechanism for stabilization of IFN-tau. This study represents the first detailed biophysical study of specific buffer-induced stabilization, resulting in shifting the equilibrium towards the native state and away form the expanded aggregation-competent species.

Solution behavior of a novel type 1 interferon, interferon-tau.

Interferon-tau (IFN-tau) is a novel cytokine that appears during fetal development of mammals. It is currently being investigated for treatment of viral infections and autoimmune diseases. In order to develop a commercial product, a stable formulation will need to be identified. In this study, the solution behavior of IFN-tau was studied using a variety of biophysical methods. The overall structure of IFN-tau is well defined, with the polypeptide chain folding into a four-helix bundle structure, much like other type 1 interferons. However, its solution behavior has not been characterized. The globular structure has a free energy of unfolding of approximately 4 kcal/mole at room temperature. IFN-tau was found to remain monomeric upon increasing the protein concentration, even up to 60 mg/mL. The overall structure of IFN-tau is maintained across a pH range of 2-8, but is significantly altered in the presence of nonaqueous solvents. However, IFN-tau appears to refold efficiently when diluted into an aqueous medium from a nonaqueous solution. This behavior allows the protein to be formulated in low water content formulations suitable for use in capsules.

Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation.

The objectives of this work were to determine the effects of nonionic surfactants (Tween 20 and Tween 80) on agitation-induced aggregation of the recombinant fusion protein, Albutropintrade mark (human growth hormone genetically fused to human albumin), and to characterize the binding interactions between the surfactants and the protein. Knowing the binding stoichiometry would allow a rational choice of surfactant concentration to protect the protein from surface-induced aggregation. Fluorescence spectroscopy and isothermal titration calorimetry (ITC) were employed to study Albutropin surfactant binding. Albutropin was agitated at 25 +/- 2 degrees C to induce aggregation, and samples were taken during a 96-h incubation. Size-exclusion chromatography (SEC-HPLC) (HPLC, high-performance liquid chromatography) was used to detect and quantify the extent of protein aggregation. The effect of surfactants on the protein's free energy of unfolding was determined using guanidine HCl as a denaturant. Tween 20 and Tween 80 had saturable binding to Albutropin with a molar binding stoichiometry of 10:1 and 9:1 (surfactant:protein), respectively. Binding of the surfactants to Albutropin increased the free energy of unfolding by over 1 and 0.6 kcal/mol, respectively. In protein samples that were agitated in the absence of surfactant, soluble aggregates were detected within 24 h, and there was almost complete loss of monomer to soluble aggregates by the end of the 96-h experiment. At the molar binding stoichiometry, Tween 20 and Tween 80 prevented the formation of soluble aggregates, even though the concentrations of surfactants were well below their critical micelle concentrations (CMC). Tween 20 and Tween 80 protected Albutropin against agitation-induced aggregation, even at concentrations below the CMC. Equilibrium unfolding data indicate that Tween confer protection by increasing the free energy of unfolding of Albutropin.