protein formulation，biopharmaceuticals

Even though arginine hydrochloride has been recognized as a protein aggregation suppressor in the biopharmaceutical industry, its use has been questioned due to decreasing transition unfolding temperatures (Tm ). Four compounds were designed to enhance the role of arginine by changing the length of the carbon chain with removal or N-acetylation of α-amino group. Biophysical properties were observed by differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow imaging (FI)...

Recently, biologics including peptides, proteins, antibodies, and nucleic acids have attracted interest as drug candidates for new modalities, since these compounds can act on target molecules that are not be affected by conventional drugs with a small molecular weight to promote greater selectivity, potency, and safety. Generally, to administer biologics, parenteral routes like intravenous and intramuscular injections have been mainly selected due to their poor oral absorbability and stability in the gastrointestinal tract, which can adversely affect patient compliance...

Discrimination between potentially immunogenic protein aggregates and harmless pharmaceutical components, like silicone oil, is critical for drug development. Flow imaging techniques allow to measure and, in principle, classify subvisible particles in protein therapeutics. However, automated approaches for silicone oil discrimination are still lacking robustness in terms of accuracy and transferability. In this work, we present an image-based filter that can reliably identify silicone oil particles in protein therapeutics across a wide range of parenteral products...

Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein-excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients...

Proteins and peptides have emerged in recent years to treat a wide range of multifaceted diseases such as cancer, diabetes and inflammation. The emergence of polypeptides has yielded advancements in the fields of biopharmaceutical production and formulation. Polypeptides often display poor pharmacokinetics, limited permeability across biological barriers, suboptimal biodistribution, and some proclivity for immunogenicity. Frequent administration of polypeptides is generally required to maintain adequate therapeutic levels, which can limit efficacy and compliance while increasing adverse reactions...

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stress are polysorbate20 (PS20) and polysorbate 80 (PS80). In recent years, polysorbate degradation in biopharmaceutical formulations has been observed. Polysorbate (PS) is mainly composed of sorbitan and isosorbide fatty acid (FA) esters, varying in their FA composition. Especially hydrolysis, which can be induced chemically as well as enzymatically, leads to the release of FAs from PS. These FAs are poorly soluble in aqueous buffer systems due to their hydrophobic nature and therefore prone to precipitation and particle formation...

Dropping during shipping and handling of liquid biopharmaceutical formulations has long been known to cause protein degradation and aggregation. On the other hand, accidental dropping of freeze-dried protein formulations is generally considered not a major issue for biopharmaceutical quality. Reports of stability and especially the underling degradation mechanism(s) during shipping and handling of freeze-dried protein formulations were rarely seen in literature. In this manuscript, we report an interesting phenomenon in which repeated dropping of freeze-dried monoclonal antibody X (mAb-X) formulation powder resulted in significant protein sub-visible particles (SbVPs) in the reconstituted liquid as determined by the sensitive particle analyzing technique micro-flow imaging (MFI)...

Surfactants are essential components in protein formulations protecting them against interfacial stress. One of the current industry-wide challenges is enzymatic degradation of parenteral surfactants such as polysorbate 20 (PS20) and polysorbate 80, which leads to the accumulation of free fatty acids (FFAs) potentially forming visible particles over the drug product shelf-life. While the concentration of FFAs can be quantified, the time point of particle formation remains unpredictable. In this work, we studied the influence of glass leachables as nucleation factors for FFA particle formation...

Host cell proteins (HCPs) are a major class of bioprocess-related impurities generated by the host organism and are generally present at low levels in purified biopharmaceutical products. The monitoring of these impurities is identified as an important critical quality attribute of monoclonal antibody (mAb) formulations not only due to the potential risk for the product stability and efficacy but also concerns linked to the immunogenicity of some of them. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), mass spectrometry (MS)-based approaches have been emerging as powerful and promising alternatives providing qualitative and quantitative information...

Polysorbates and Poloxamer 188 constitute the most common surfactants used in biopharmaceutical formulations owing to their excellent protein-stabilizing properties and good safety profiles. In recent years, however, a vast number of reports concerning potential risk factors closely related with their applications, such as the accumulation of degradation products, their inherent heterogeneity and adsorption effects of proteins at silicon/oil interfaces have drawn the focus to potential alternatives. Apart from tedious efforts to evaluate new excipient candidates, the use of mixed formulations leveraging combinations of well-established surfactants appears to be a promising approach to eliminate or, at least, minimize and postpone adverse effects associated with the single compounds...

Polysorbate 20 (PS20) is a commonly used surfactant in biopharmaceutical formulations. It is a heterogeneous surfactant containing a distribution of fatty acid esters, which are subject to hydrolytic degradation, generating free fatty acids (FFAs). The FFAs can form visible or subvisible particles in drug product on stability. A previous FFA solubility model, developed by our group, predicts solubility limits for the three most prevalent FFA degradation products of PS20: lauric, myristic, and palmitic acid...

Circular dichroism (CD) spectra of biopharmaceutical protein, or protein conjugate, products contain information about their secondary and tertiary structures, which can answer to increasing regulatory interest in demonstrating consistent higher order structures of production batches. Widespread routine use of CD in a regulatory environment requires objective, statistically based, and validated methods to analyse and compare spectra against product specifications. Correlation approaches to compare spectra, developed and tested on monoclonal antibodies, are here used to assess the consistency of Hib PRP-CRM197 glycoconjugate immunogen batches, by analysis of historical data sets...

Bottles and carboys are used for frozen storage and transport of biopharmaceutical formulations under a wide range of conditions. The quality of freezing and thawing in these systems has been questioned due to the formation of heterogeneous ice structures and deformation of containers. This work shows that during freezing of bulk protein solutions, the liquid at the air-liquid interface freezes first, forming an ice crust and enclosing the liquid phase. As the enclosed liquid freezes, internal pressure rises, pushing the liquid phase through the porous ice crust towards the air interface, leading to interfacial stress and protein aggregation...

Polysorbates are amphiphilic, non-ionic surfactants, and they represent one of the key components of biopharmaceuticals. They serve as stabilisers, and their degradation can cause particle formation, which has been an industry-wide issue over the past decade. To determine the influence of the buffers most frequently used in biopharmaceuticals on polysorbate degradation, an accelerated stability study was carried out using placebo formulations containing 0.02% polysorbates and 20 mM buffers (pH 5.5, 6.5). These included histidine chloride, sodium citrate, sodium succinate and sodium phosphate buffers...

Controlling the viscosity of concentrated protein solutions - usually reducing - is an open challenge, with major recent relevance in protein formulations for biopharmaceutical, medical, food, and other applications. The addition of viscosity-reducing additives generally not only changes the viscosity of the protein solutions but also the actual secondary/tertiary structure of the proteins, which is usually highly undesirable, and can be even toxic in systems, such as for biopharmaceutical applications. Therefore, it is of major importance to be able to establish control over the combination of viscosity-affecting additives and adequate protein stability, usually at high protein concentrations...

Super Refined™ and Tween™ 20 HP polysorbate 20 (PS20) are two commercially available compendial grades of PS20 frequently used in biopharmaceutical formulations as protein stabilizing surfactants. PS20 degradation has been a major concern recently for potentially limiting drug product shelf life due to free fatty acid particle formation. This work is a side-by-side comparison of SR and HP PS20 in terms of PS20 degradation, particle formation and protein stability. The use of SR grade PS20 results in higher levels of oxidative PS20 degradation, protein oxidation, peroxides and protein aggregation, and therefore requires addition of methionine as an antioxidant to mitigate these issues...

Monitoring protein particles is increasingly emphasized in the development of biopharmaceuticals due to potential immunogenicity. Accurate quantitation of protein particles is complicated by silicone oil droplets, a common pharmaceutical component in pre-filled syringes. Though silicone oil is typically regarded as harmless, numerous reports have indicated protein adsorption may render these particles with immunostimulatory properties. Imaging flow cytometry (IFC) is an emerging pharmaceutical method capable of capturing high-resolution brightfield and fluorescence imagery from samples in suspension...

Therapeutic monoclonal antibodies (mAbs) are highly complex proteins that must be exhaustively characterized according to the regulatory authorities' recommendations. MAbs display micro-heterogeneity mainly due to their post-translational modifications, but also to their susceptibility to chemical and physical degradations. Among these degradations, aggregation is quite frequent, initiated by protein denaturation and then dimer formation. Here, we investigated the nature and structure of the high molecular weight species (HMW) present at less than 1% in an unstressed formulated roledumab biopharmaceutical, as a model of high purity mAb...

Modern therapy of metabolic, neurodegenerative, inflammation, or cancer diseases is recently based on an immunotherapeutic approach. The peptide conjugates represent innovative and effective therapeutics that are better tolerated and are much more specific than small molecule-based medicines. The nature and manufacturing process of these therapeutics make their analysis very challenging. Here, two robust analytical methods based on an on-line combination of ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) and capillary electrophoresis with tandem mass spectrometry (CE-MS/MS) were developed for fast determination of immunogenic synthetic peptide (peptide sequence CADNLHKVVGQST) in a conjugate with bovine serum albumin (BSA) as a carrier protein and is a peptide, conjugate formulated with a vaccine adjuvant - Alhydrogel® 2 %...

Exosomes are small nanoparticles secreted by almost all cells and have a well-known role in intercellular communication. They are found in different body fluids and can also be isolated from cell culture media. They contain a natural cargo including various protein and nucleic acid molecules originated from their donor cells. In recent years, exosomes have emerged as a desired drug delivery system. They are believed to provide a targeted delivery of drug molecules, supplemented with their natural function. Furthermore, they have a membranous structure similar to liposomes, and that motivated researchers to apply their previous experience of drug loading into liposomes for exosomes...