protein formulations
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UV and ambient light-induced modifications and related degradation of therapeutic proteins are observed during manufacturing and storage. Therefore, to ensure product quality, protein formulations need to be analyzed with respect to photo-degradation processes and eventually protected from light exposure. This task usually demands the application and combination of various analytical methods. This review addresses analytical aspects of investigating photo-oxidation products and related mediators such as reactive oxygen species generated via UV and ambient light with well-established and novel techniques.

A fundamental step in developing a protein drug is the selection of a stable storage formulation that ensures efficacy of the drug and inhibits physiochemical degradation or aggregation. Here, we designed and evaluated a general workflow for screening of protein formulations based on small-angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling, temperature control, and fast data analysis and provides protein particle interaction information. SAXS, together with different methods including turbidity analysis, dynamic light scattering (DLS), and SDS-PAGE measurements, were used to obtain different parameters to provide high throughput screenings. Using a set of model proteins and biopharmaceuticals, we show that SAXS is complementary to dynamic light scattering (DLS), which is widely used in biopharmaceutical research and industry. We found that, compared to DLS, SAXS can provide a more sensitive measure for protein particle interactions, such as protein aggregation and repulsion. Moreover, we show that SAXS is compatible with a broader range of buffers, excipients, and protein concentrations and that in situ SAXS provides a sensitive measure for long-term protein stability. This workflow can enable future high-throughput analysis of proteins and biopharmaceuticals and can be integrated with well-established complementary physicochemical analysis pipelines in (biopharmaceutical) research and industry.

Abstract Since 1986 when we first witnessed the approval of monoclonal antibody (mAb) Orthoclone OKT3 by the US FDA, FDA has approved 103 therapeutic antibody drugs in the past 35 years for marketing. Thirty four (34) of these 103 therapeutic antibody drugs (accounting for one third of the total FDA approved antibody therapeutics) are formulated with high protein concentration (100 mg/mL or above). These 34 high concentration antibodies are the focus of this article. The dosage forms of these 34 antibodies are analyzed and discussed in this article. The highest protein concentration of these approved mAbs is 200 mg/mL. The dominant administration route is subcutaneous (76%). Our analysis indicates that it may be rational to implement a platform formulation containing polysorbate, histidine and sucrose to accelerate high concentration formulation development for antibody drugs. The top players/sponsors of high concentration formulation are identified as Roche including its subsidiaries Genentech and Chugai, Novartis, Sanofi, Amgen, GSK, Johnson & Johnson including its subsidiary Janssen, and Regeneron. The FDA approval numbers are significantly increased since 2015 which account for 76% of the total approval number, i.e., 26 out of 34 highly concentrated antibodies. Thus, we believe that the high concentration formulations of antibody drugs will be the future trend of therapeutic antibody formulation development, regardless of the challenges of highly concentrated protein formulations.

Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180–250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.

Introduction. New legume-based protein preparations are an excellent alternative to polymers of animal origin and can eliminate the protein deficiency in the diet of humans and animals. In this respect, the raw material base of common leguminous crops has to be thoroughly analyzed in order to develop new technological schemes for novel protein formulations. Study objects and methods. The present research compared modern trends in the production, properties, and safety of food and feed protein preparations based on peas and chickpeas. It involved such standard methods as data systematization and analysis of literary sources. Results and discussion. The leguminous agriculture in Russia is stable enough to produce food and feed protein preparations from peas and chickpeas with the maximum preservation of biological value, composition, and properties. Peas and chickpeas have a high biological value and are rich in polypeptides, fiber, minerals, vitamins, antioxidants, etc., which are lost during processing. By-products of protein production can be processed using biosynthetic transformation with various types of fungal and/or bacterial enzymes, as well as physical and/or physicochemical methods, to obtain feed or food products with an appropriate yield. A synthesis with enzymes or microorganisms can result in functional foods and feeds fortified with minerals, vitamins, fatty acids, and antioxidants.

Microviridins and other ω-ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAsp-containing protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.

The perming of hair is a common styling procedure with negative impact on the overall properties of the hair fibers. Usually, this process uses harsh chemicals to promote the disruption of disulfide bonds and the formation of new bonds to change the shape of hair. Here, we explored bovine serum albumin (BSA), silk fibroin (SF), keratin and two fusion recombinant proteins (KP-UM and KP-Cryst) as new perming agents. A phosphate buffer prepared at different pH values (5, 7 and 9) was used to apply the proteins to virgin Asian hair, and a hot BaByliss was used to curl the hair fibers. To assess the potential of the protein formulations for hair styling, the perming efficiency and the perming resistance to wash were measured. Furthermore, the fiber water content was evaluated to assess if the proteins protected the hair during the styling process. Despite all of the proteins being able to assist in the curling of Asian hair, the best perming efficiency and perming resistance to wash results were observed for BSA and keratin. These proteins showed perming efficiency values close to that measured for a commercial perming product (chemical method), particularly at pH 5 and 9. The increase in the hair’s internal and external water contents revealed a protective effect provided by the proteins during the application of heat in the styling procedure. This study shows the potential of proteins to be used in the development of new eco-friendly hair styling products.