Protein Formulations Research Articles

Hierarchical clustering of therapeutic proteins based on agitation-induced aggregation propensity and its relation to physicochemical parameters.

Hierarchical clustering of therapeutic proteins based on agitation-induced aggregation propensity and its relation to physicochemical parameters.

Impact of Sugar Molecular Weight on the Miscibility and Stability of Lyophilized and Spray-Dried Protein Formulations.

Poor stability of biological products such as proteins is a major challenge facing the biopharmaceutical industry. Poor stability is usually mitigated by formulating these products in the solid state, employing sugars as stabilizers. Several studies have pointed out the superior stabilizing ability of disaccharides, including sucrose and trehalose, as compared to polysaccharides such as dextrans. The aim of this study was to investigate the impact of excipient molecular weight on miscibility with a model protein, bovine serum albumin (BSA). Aqueous solutions containing a binary combination of a sugar-based stabilizer and BSA were dried using different methods (air drying to form films, spray drying, and lyophilization). The stabilizers tested varied in molecular weight and were dextran 6, 70, or 2000 kDa, hydroxypropyl methyl cellulose (HPMC), and trehalose. Miscibility was evaluated using a variety of techniques including confocal fluorescence microcopy, infrared and Raman microscopy, and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. The stability of BSA in dried mixtures subjected to accelerated storage conditions was also measured. BSA was more stable in the presence of dextran 2000 kDa compared to dextran 70 and 6 kDa, while stability was highest in trehalose and lowest in HPMC. From ssNMR spectroscopy, BSA-Dex 2000 kDa and BSA-trehalose were miscible over 20 and 5 nm length scales, BSA-Dex 6 kDa was miscible over a 20 nm length scale and phase-separated over a 5 nm length scale, while BSA-Dex 70 kDa and BSA-HPMC were phase-separated over both length scales. It was postulated that for dextran, the size of the polysaccharide relative to the size of the protein determined the extent of the system miscibility and stability. A smaller or similar polysaccharide size compared to that of the protein, as in the case of BSA-Dex 6 kDa and BSA-Dex 70 kDa, leads to depletion-induced phase separation. A much larger polysaccharide size compared to that of the protein allows the protein molecules to be trapped within a polysaccharide mesh, resulting in a miscible system. This study suggests that the impact of the relative size of the stabilizer and protein on miscibility is more complex than previously considered.

Role of hydrogen bonding and water clusters in deamidation of peptide in glycerol-water solutions.

Role of hydrogen bonding and water clusters in deamidation of peptide in glycerol-water solutions.

Effects of salts, buffers and sucrose on protein-protein attractive and repulsive interactions.

Effects of salts, buffers and sucrose on protein-protein attractive and repulsive interactions.

An exploration of applied plant-based protein formulations to shift farmers towards sustainable diets: A South African Perspective

An exploration of applied plant-based protein formulations to shift farmers towards sustainable diets: A South African Perspective

Controlled release of MIF siRNA and GDNF protein from a photocurable scaffold efficiently repairs spinal cord injury.

Compared with traditional treatment strategies, siRNA-based gene therapy combines with protein therapy to offer a new strategy for spinal cord injury (SCI). The siRNA and protein therapy are limited by the large and deep lesion site and local co-delivery vectors. However, the photocurable scaffold has the properties of injectable, flexible, and biodegradable, which provide a potential formulation for siRNA and protein combined therapy. Here, a photocurable lipid nanoparticle gel (PLNG) scaffold is designed for efficiently sustained and controlled release of the macrophage migration-inhibitory factor (MIF) targeted siRNA and co-delivery of GDNF protein for SCI. The GDNF is chemically modified in the scaffold and the prepared GDNF-PLNG/siRNA scaffold is injectable with easily photocured. This formulation can inhibit inflammation by promoting macrophage M2 polarization and effectively promote primary neuron axon growth. After locally administered with GDNF-PLNG/siMIF scaffold to SCI mice, the scaffold promoted neuron regeneration by upregulation of neuron cytokine production and inhibited inflammation through the downregulation immune pathway. With the interaction mechanism of GDNF and MIF siRNA, GDNF-PLNG/siMIF scaffold increases the collagen and integrin expression to promote spinal cord repairing and significantly improve motor function, so that scaffold is a potential candidate gene formulation applied to clinical SCI treatment.

Higher concentration of trehalose dihydrate stabilizes recombinant IgG1 under forced stress conditions.

Higher concentration of trehalose dihydrate stabilizes recombinant IgG1 under forced stress conditions.

The effect of glass container surface silanol density on monoclonal antibody formulation stability after application of mechanical shock.

The effect of glass container surface silanol density on monoclonal antibody formulation stability after application of mechanical shock.

A review of polysorbate quantification and its degradation analysis by liquid chromatography coupled with charged aerosol detection.

A review of polysorbate quantification and its degradation analysis by liquid chromatography coupled with charged aerosol detection.

Representative training data sets are critical for accurate machine-learning classification of microscopy images of particles formed by lipase-catalyzed polysorbate hydrolysis.

Representative training data sets are critical for accurate machine-learning classification of microscopy images of particles formed by lipase-catalyzed polysorbate hydrolysis.

Turning Diverse Analytical Data into Actionable Knowledge for Enzymatically Driven Polysorbate Degradation Risk Assessment and Control in Biotherapeutic Protein Formulations

Turning Diverse Analytical Data into Actionable Knowledge for Enzymatically Driven Polysorbate Degradation Risk Assessment and Control in Biotherapeutic Protein Formulations

A Precipitation-Based Process to Generate a Solid Formulation of a Therapeutic Monoclonal Antibody: An Alternative to Lyophilization

Lyophilization, or freeze-drying, is the default technique for the manufacture of solid-state formulations of therapeutic proteins. This established method offers several advantages, including improved product stability by minimizing chemical degradation, reduced storage requirements through water removal, and elimination of cold chain dependence. However, the lyophilization process itself presents limitations. It is a lengthy, batch-based operation, potentially leading to product inconsistencies and high manufacturing costs. Additionally, some proteins are susceptible to structural alterations during the freezing step, impacting their biological activity. This paper presents an alternative approach based on the co-precipitation of protein and excipients using an organic solvent. We explore the impact of various processing parameters on the viability of the formulation. We also provide an extensive characterization of proteins reconstituted from precipitated formulations and compare protein stability in solution and in lyophilized and precipitated solid formulations under long-term, accelerated, and stressed storage conditions.

Formulation Factors Affecting the Formation of Visible-Bubbles During the Reconstitution Process of Freeze-Dried Etanercept Formulations: Protein Concentration, Stabilizers, and Surfactants.

Freeze drying is one of the common methods to extend the long-term stability of biologicals. Biological products in solid form have the advantages of convenient transportation and stable long-term storage. However, long reconstitution time and extensive visible bubbles are frequently generated during the reconstitution process for many freeze-dried protein formulations, which can potentially affect the management efficiency of staff, patient compliance, and product quality. The reconstitution time has been extensively studied, but the influence of the formulations on the formation of visible bubbles is often overlooked. This paper investigated the effect of freeze-drying formulation factors (i.e., protein concentrations, surfactant concentrations, and sucrose/mannitol compositions) on product stability and visible bubbles generated during reconstitution of freeze-dried etanercept formulations. The generating and breakup mechanisms of visible bubbles were detected via internal microstructure of cake, surface tension, and viscosity measurement. Under the same protein concentration, the formulation of mannitol mixed with sucrose in a weight ratio of 4:1 produces fewer visible bubbles during the reconstitution process compared to the formulation of sucrose with the same total mass. This has been proven to be due to the large number of smaller radius pores distributed in the pores of the freeze-dried cake of the former, while the average internal structure pores of the latter are much larger than those of the former. As an amorphous stabilizer, sucrose can ensure the long-term stability of protein and greatly reduce the generation and maintenance of foams in the reconstitution process, making it a more robust excipient for freeze-dried protein formulations.

Rapid Development of High Concentration Protein Formulation Driven by High-Throughput Technologies.

High concentration protein formulation (HCPF) development needs to balance protein stability attributes such as conformational/colloidal stability, chemical stability, and solution properties such as viscosity and osmolality. A three-phase design is established in this work. In Phase 1, conformational and colloidal stability are measured by 384-well-based high-throughput (HT) biophysical screening while viscosity reduction screening is performed with HT viscosity screening. Collectively, the biophysical and viscosity screening data are leveraged to design the phase 2 of short-term stability study, executed using 96-well plates under thermal and freeze/thaw stresses. In phase 2, samples are analyzed by stability-indicating assays and processed with pair-wise Student's t-test analyses to choose the final formulations. In phase 3, the final formulations are then confirmed through a one-month accelerated stability in glass vials. Using a model antibody A (mAb-A), the initial HT screening successfully established the 384-well based platform. A lead formulation was chosen from the second round based on statistical analyses and subsequently tested against the commercial formulation of mAb-A as a control. Compared to the control, the lead formulation reduced the viscosity of mAb-A by 30% and decreased subvisible particles after thermal stress by 80%. HT biophysical screening in 384-well plates was demonstrated to effectively guide the rational design of a high-throughput stability screening study using 96-well plates. This platform enables the identification of a high concentration formulation within seven weeks within the first two phases of study that strategically balance stability with solution properties, thus achieving a rapid development of HCPF.

Heat stable and intrinsically sterile liquid protein formulations

Over 80% of biologic drugs, and 90% of vaccines, require temperature-controlled conditions throughout the supply chain to minimize thermal inactivation and contamination. This cold chain is costly, requires stringent oversight, and is impractical in remote environments. Here, we report chemical dispersants that non-covalently solvate proteins within fluorous liquids to alter their thermodynamic equilibrium and reduce conformational flexibility. This generates non-aqueous, fluorine-based liquid protein formulations that biochemically rigidify protein structure to yield thermally stable biologics at extreme temperatures (up to 90 °C). These non-aqueous formulations are impervious to contamination by microorganismal pathogens, degradative enzymes, and environmental impurities, and display comparable pre-clinical pharmacokinetics and safety profiles to standard saline protein samples. As a result, we deliver a fluorochemical formulation paradigm that may limit the need for cold chain logistics of protein reagents and biopharmaceuticals.

Current Advancements on Oral Protein and Peptide Drug Delivery Approaches to Bioavailability: Extensive Review on Patents.

Protein and peptide-based drugs have greater therapeutic efficacy and potential application and lower toxicity compared to chemical entities in long-term use within optimum concentration as they are easily biodegradable due to biological origin. While oral administration is preferable, most of these substances are currently administered intravenously or subcutaneously. This is primarily due to the breakdown and poor absorption in the GI tract. Hence, ongoing research is focused on investigating absorption enhancers, enzyme inhibitors, carrier systems, and stability enhancers as potential strategies to facilitate the oral administration of proteins and peptides. Investigations have been directed towards advancing novel technologies to address gastrointestinal (GI) barriers associated with protein and peptide medications. The current review intensifies formulation and stability approaches for oral protein & peptide drug delivery systems with all significant parameters intended for patient safety. Notably, certain innovative technologies have been patented and are currently undergoing clinical trials or have already been introduced into the market. All the approaches stated for the administration of protein and peptide drugs are critically discussed, having their current status, future directions, and recent patents published in the last decades.

"The More, the Better?": The Impact of Sugar-to-Protein Molar Ratio in Freeze-Dried Monoclonal Antibody Formulations on Protein Stability.

Lyophilization is widely used to ensure the stability of protein drugs by minimizing chemical and physical degradation in the dry solid state. To this end, proteins are typically formulated with sugars that form an amorphous immobilizing matrix and stabilize hydrogen bonds replacing water molecules. The optimal amount of sugar required and protein stability at low excipient-to-protein molar ratios are not well understood. We investigated this by focusing on the physical stability of formulations, reflecting highly concentrated monoclonal antibody (mAb) lyophilizates at low sucrose to mAb ratios between 25:1 and 360:1. Additionally, the impact of different excipient types, buffer concentrations, and polysorbates was studied. The mAb stability was evaluated over up to three months at 25 and 40 °C. We investigated the "the more, the better" approach regarding excipient usage in protein formulation and the existence of a potential stabilizing threshold. Our findings show efficient monomeric content preservation even at low molar ratios, which could be explained based on the water replacement theory. We identified an exponential correlation between the sucrose to protein molar ratio and aggregate formation and found that there is no molar ratio threshold to achieve minimum stabilization. Sucrose demonstrated the best stabilization effect. Both mannitol, used as a cryoprotectant at low concentrations, and arginine reduced aggregation compared to the pure mAb formulation. The higher ionic strength of 5 mM histidine buffer enhanced protein stability compared to that of 0.1 mM histidine buffer, which was more pronounced at lower molar ratios. The addition of polysorbate 20 contributed an additional interfacial stabilizing effect, complementing the cryoprotective and lyoprotective properties of sucrose. Overall, a model could be developed to optimize the quantity of sugar required for protein stabilization and facilitate a more rational design of protein lyophilizates. The molar ratio of sugar to protein for high-concentration mAb products is limited by the acceptable tonicity, but we showed that sufficient stabilization can be achieved even at low molar ratios.

Effects of buffers on spray-freeze-dried/lyophilized high concentration protein formulations

Effects of buffers on spray-freeze-dried/lyophilized high concentration protein formulations

Investigation of Protein Therapeutics in Frozen Conditions Using DNP MAS NMR: A Study on Pembrolizumab.

The success of modern biopharmaceutical products depends on enhancing the stability of protein therapeutics. Freezing and thawing, which are common thermal stresses encountered throughout the lifecycle of drug substances, spanning protein production, formulation design, manufacturing, storage, and shipping, can impact this stability. Understanding the physicochemical and molecular behaviors of components in biological drug products at temperatures relevant to manufacturing and shipping is essential for assessing stability risks and determining appropriate storage conditions. This study focuses on the stability of high-concentration monoclonal antibody (mAb) pembrolizumab, the drug substance of Keytruda (Merck & Co., Inc., Rahway, NJ, United States), and its excipients in a frozen solution. By leveraging dynamic nuclear polarization (DNP), we achieve more than 100-fold signal enhancements in solid-state NMR (ssNMR), enabling efficient low-temperature (LT) analysis of pembrolizumab without isotopic enrichment. Through both ex situ and in situ ssNMR experiments conducted across a temperature range of 297 to 77 K, we provide insights into the stability of crystalline pembrolizumab under frozen conditions. Importantly, utilizing LT magic-angle spinning (MAS) probes allows us to study molecular dynamics in pembrolizumab from room temperature down to liquid nitrogen temperatures (<100 K). Our results demonstrate that valuable insights into protein conformation and dynamics, crystallinity, and the phase transformations of excipients during the freezing of the formulation matrix can be readily obtained for biological drug products. This study underscores the potential of LT-MAS ssNMR and DNP techniques for analyzing protein therapeutics and vaccines in frozen solutions.

Evaluating the influence of the initial high molecular weight level on monoclonal antibody particle formation kinetics using a short-term chemical stress study

Evaluating the influence of the initial high molecular weight level on monoclonal antibody particle formation kinetics using a short-term chemical stress study