Addressing common challenges of biotherapeutic protein peptide mapping using recombinant trypsin.

Peptide mapping is the key method for characterization of primary structure of biotherapeutic proteins. This method relies on digestion of proteins into peptides that are then analyzed for amino acid sequence and post-translational modifications. Owing to its high activity and cleavage specificity, trypsin is the protease of choice for peptide mapping. In this study, we investigated critical requirements of peptide mapping and how trypsin affects these requirements. We found that the commonly used MS-grade trypsins contained non-specific, chymotryptic-like cleavage activity causing generation of semi-tryptic peptides and degradation of tryptic-specific peptides. Furthermore, MS-grade trypsins contained pre-existing autoproteolytic peptides and, moreover, additional autoproteolytic peptides were resulting from prominent autoproteolysis during digestion. In our long-standing quest to improve trypsin performance, we developed novel recombinant trypsin and evaluated whether it could address major trypsin drawbacks in peptide mapping. The study showed that the novel trypsin was free of detectable non-specific cleavage activity, had negligible level of autoproteolysis and maintained high activity over the course of digestion reaction. Taking advantage of the novel trypsin advanced properties, especially high cleavage specificity, we established the application for use of large trypsin quantities to digest proteolytically resistant protein sites without negative side effects. We also tested trypsin/Lys-C mix comprising the novel trypsin and showed elimination of non-specific cleavages observed in the digests with the commonly used trypsins. In addition, the improved features of the novel trypsin allowed us to establish the method for accurate and efficient non-enzymatic PTM analysis in biotherapeutic proteins.

Human Pineal Gland Involutionary Process: New Findings.

In this work, we report preliminary results about the involution of the human pineal gland involution. The detailed analysis of pineal structure was done on autopsy material of 77 persons in age 27-96 using x-ray phase-contrast tomography, histology, and immunohistochemistry. Our study suggests that the pineal gland alteration in older adults may be more profound than has been reported to date. We identified and described a new form of pineal gland involution that eventually led to the total degradation of the pineal gland. To our knowledge, this study is the first to report on the complete replacement of pineal gland parenchyma with connective tissue in older adults.

Physicochemical characterization and functionality comparison of Humira®(adalimumab), Remicade®(infliximab) and Simponi Aria®(golimumab).

FDA-approved anti-TNFα biopharmaceuticals are successful in treating a range of autoimmune diseases. However, not all anti-TNFα products are identical in their patient outcomes, suggesting that there may be product-specific differences stemming from protein structural differences, doses and routes of administration. In this work, we focus only on structural and functional differences across three full-length anti-TNFα mAbs (Humira®, Remicade®, and Simponi Aria®) to better understand the implications of such differences on the products' efficacy. For structural characterization, we quantified N-glycans using mass spectrometry and fluorescence labeling. From these studies, we observed that Remicade® had the highest percent of afucosylated glycans (15.5 ± 1.3 %) and the largest number of unique glycans, 28. While Humira® had the fewest unique glycans, 15, and 11.4 ± 0.8 % of afucosylated, high-mannose glycans. For the functional studies we tested TNFα binding via ELISA, FcγRIIIa binding via AlphaLISA and effector function using an ADCC bioreporter assay. Humira® had a significantly lower EC50 (1.9 ± 0.1 pM) for ELISA and IC50 (10.5 ± 1.1 nM) for AlphaLISA, suggesting that Humira® has higher TNFα and FcγRIIIa binding affinity than Remicade® and Simponi Aria®. Humira® was also the most potent in the bioreporter assay with an EC50 value of 0.55 ± 0.03 nM compared to Remicade® (0.64 ± 0.04 nM) and Simponi Aria® (0.67 ± 0.03 nM). This comparison is significant as it highlights functional differences between mAbs with shared mechanisms of action when examined in a single laboratory and under one set of conditions.

Streamlining the Characterization of Disulfide Bond Shuffling and Protein Degradation in IgG1 Biopharmaceuticals Under Native and Stressed Conditions.

Post translational modifications (PTMs) have been shown to negatively impact protein efficacy and safety by altering its native conformation, stability, target binding and/or pharmacokinetics. One PTM in particular, shuffled disulfide bonds, has been linked to decreased potency and increased immunogenicity of protein therapeutics. In an effort to gain more insights into the effects of shuffled disulfide bonds on protein therapeutics' safety and efficacy, we designed and further optimized a semi-automated LC-MS/MS method for disulfide bond characterization on two IgG1 protein therapeutics-rituximab and bevacizumab. We also compared originator vs. biosimilar versions of the two therapeutics to determine if there were notable variations in the disulfide shuffling and overall degradation between originator and biosimilar drug products. From our resulting data, we noticed differences in how the two proteins degraded. Bevacizumab had a general upward trend in shuffled disulfide bond levels over the course of a 4-week incubation (0.58 ± 0.08% to 1.46 ± 1.10% for originator) whereas rituximab maintained similar levels throughout the incubation (0.24 ± 0.21% to 0.51 ± 0.11% for originator). When we measured degradation by SEC and SDS-PAGE, we observed trends that correlated with the LC-MS/MS data. Across all methods, we observed that the originator and biosimilar drugs performed similarly. The results from this study will help provide groundwork for comparative disulfide shuffling analysis by LC-MS/MS and standard analytical methodology implementation for the development and regulatory approval of biosimilars.

Multifaceted assessment of rituximab biosimilarity: The impact of glycan microheterogeneity on Fc function.

Biosimilars are poised to reduce prices and increase patient access to expensive, but highly effective biologic products. However, questions still remain about the degree of similarity and scarcity of information on biosimilar products from outside of the US/EU in the public domain. Thus, as an independent entity, we performed a comparative analysis between the innovator, Rituxan® (manufactured by Genentech/Roche), and a Russian rituximab biosimilar, Acellbia® (manufactured by Biocad). We evaluated biosimilarity of these two products by a variety of state-of-the-art analytical mass spectrometry techniques, including tandem MS mapping, HX-MS, IM-MS, and intact MS. Both were found to be generally similar regarding primary and higher order structure, though differences were identified in terms of glycoform distribution levels of C-terminal Lys, N-terminal pyroGlu, charge variants and soluble aggregates. Notably, we confirmed that the biosimilar had a higher level of afucosylated glycans, resulting in a stronger FcγIIIa binding affinity and increased ADCC activity. Taken together, our work provides a comprehensive comparison of Rituxan® and Acellbia®.

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping.

mAbs undergo several post-translational modifications, including the formation of succinimide from the deamidation of asparagine or the isomerization of aspartic acid. Because of the potential impact of succinimide formation on the biological activity of mAbs, detection and quantification of this species is a key area of interest for the pharmaceutical industry. However, studies assessing succinimide stability have been limited, and methods developed to monitor succinimide are either product specific or not robust. Here, we report the development of a platform low-pH peptide-mapping method using a combination of low-pH-resistant Lys-C and modified trypsin to maintain succinimide stability, eliminate deamidation assay artifact, and achieve efficient mAb digestion equivalent to conventional tryptic peptide-mapping method under alkaline condition. Using this method, succinimide stability in serum was accurately assessed in vitro study and the half-life was determined to be 1.5 days. With potential patient exposure to succinimide intermediate, a reliable method was developed to measure site-specific deamidation and succinimide intermediate. Coupled with a single quadrupole mass detector, our method was automated from digestion to data processing and applicable in a good manufacturing practice environment. The method was fully qualified to demonstrate accuracy, precision, linearity, and robustness.

Characterization and quantification of succinimide using peptide mapping under low-pH conditions and hydrophobic interaction chromatography.

Characterization of asparagine deamidation and aspartic acid isomerization is an important aspect of biotherapeutic protein analysis due to the potential negative effect of these modifications on drug efficacy and stability. Succinimide has long been known to be an intermediate product of asparagine deamidation and aspartic acid isomerization, but despite the key role of succinimide in these reactions, its analysis remains challenging due to its instability. We have developed a paradigm in which two interlinked analytical methods are used to develop an optimized approach to analyze succinimide. In the first method, low-pH protein digestion is used for detailed characterization of succinimide with peptide mapping. At low pH, succinimide is stable and can be analyzed with accurate mass measurements and tandem mass spectrometry to confirm its identity and localize its modification site. These results are then used to establish a hydrophobic interaction chromatography (HIC)-based method that can be used for release and stability studies. In this method, unmodified protein, deamidated products, and succinimide are well separated and quantified. Good correlation was obtained between the data from low-pH protein digestion-based peptide mapping and the HIC-based method. Method qualification showed that the HIC-based method is robust, accurate, and precise and has excellent linearity.

Developmental dynamics of prepiriform cortex in prenatal human ontogenesis.

The prepiriform cortex is a part of the phylogenetically oldest pallial division (paleocortex) representing the primary olfactory cortex. While olfactory centers in laboratory animals have been extensively investigated, the developmental timetable of the human prepiriform area is poorly understood. Thus, in the present study we aim to examine the prepiriform cortex in human fetuses from eight postconceptional weeks to birth. Based on cytoarchitecture and immunohistochemistry analysis (NeuN-, SYP-, NSE-, TH-, GFAP-, MBP-) four main periods of the prepiriform cortex fetal development are suggested: the beginning of prefetal stage (the eighth week from conception), the period from the ending of prefetal stage (9-12 postconceptional weeks) to 17 weeks of gestation, 18-27 weeks of gestation and the late fetal period (29-40 gestational weeks). We found that the initial layer differentiation took place before the ninthtenth weeks from conception and by ten weeks the paleocortical plate of the prepiriform cortex was shaped. Both total cell density and NeuN-immunoreactive cell density peaked in the early fetuses and started to decrease after 17 gestational weeks, attaining intermediate values at 18-27 weeks and becoming significantly lower in the late fetuses. In contrast, the NeuN-immunoreactive cell ratio gradually increased over the whole examined period. The prepiriform cortex was defined as approaches the state at birth at 30 gestational weeks. The same developmental periods were observed with SYP- and NSE-assays. No significant distribution of TH immunoreactivity was described in the prepiriform cortex of human fetuses. The prior paleocortex development was demonstrated using glial markers: GFAPimmunoreactivity appeared in the prepiriform cortex at the middle of the early fetal period, ahead of the neocortex and insular cortex. The earlier rates of GFAP-immunoreactivity expansion in the prepiriform cortex, as compared to other pallial regions, persisted in the later fetuses. The first MBP-immunoreactive fibres within pallium were detected in the lateral olfactory tract at 30 weeks. Therefore, the prepiriform cortex approaches a level of maturation similar to that at birth already at the beginning of the late fetal period and matures prior to other pallial regions.

Biosimilarity under stress: A forced degradation study of Remicade® and Remsima™.

Remsima™ (infliximab) is the first biosimilar monoclonal antibody (mAb) approved by the European Medical Agency and the US Food and Drug Administration. Remsima™ is highly similar to its reference product, Remicade®, with identical formulation components. The 2 products, however, are not identical; Remsima™ has higher levels of soluble aggregates, C-terminal lysine truncation, and fucosylated glycans. To understand if these attribute differences could be amplified during forced degradation, solutions and lyophilized powders of the 2 products were subjected to stress at elevated temperature (40-60°C) and humidity (dry-97% relative humidity). Stress-induced aggregation and degradation profiles were similar for the 2 products and resulted in loss of infliximab binding to tumor necrosis factor and FcγRIIIa. Appearances of protein aggregates and hydrolysis products were time- and humidity-dependent, with similar degradation rates observed for the reference and biosimilar products. Protein powder incubations at 40°C/97% relative humidity resulted in partial mAb unfolding and increased asparagine deamidation. Minor differences in heat capacity, fluorescence, levels of subvisible particulates, deamidation and protein fragments were observed in the 2 stressed products, but these differences were not statistically significant. The protein solution instability at 60°C, although quite significant, was also similar for both products. Despite the small initial analytical differences, Remicade® and Remsima™ displayed similar degradation mechanisms and kinetics. Thus, our results show that the 2 products are highly similar and infliximab's primary sequence largely defines their protein instabilities compared with the limited influence of small initial purity and glycosylation differences in the 2 products.

A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima.

In April 2016, the Food and Drug Administration approved the first biosimilar monoclonal antibody (mAb), Inflectra/Remsima (Celltrion), based off the original product Remicade (infliximab, Janssen). Biosimilars promise significant cost savings for patients, but the unavoidable differences between innovator and copycat biologics raise questions regarding product interchangeability. In this study, Remicade and Remsima were examined by native mass spectrometry, ion mobility, and quantitative peptide mapping. The levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. We found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact. Importantly, we identified more than 25 glycoforms for each product and observed glycoform population differences, with afucosylated glycans accounting for 19.7% of Remicade and 13.2% of Remsima glycoforms, which translated into a 2-fold reduction in the level of FcγIIIa receptor binding for Remsima. While this difference was acknowledged in Remsima regulatory filings, our glycoform analysis and receptor binding results appear to be somewhat different from the published values, likely because of methodological differences between laboratories and improved glycoform identification by our laboratory using a peptide map-based method. Our mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. We have demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.

The mummified brain of a pleistocene woolly mammoth (Mammuthus primigenius) compared with the brain of the extant African elephant (Loxodonta africana).

This study presents the results of an examination of the mummified brain of a pleistocene woolly mammoth (Mammuthus primigenius) recovered from the Yakutian permafrost in Siberia, Russia. This unique specimen (from 39,440-38,850 years BP) provides the rare opportunity to compare the brain morphology of this extinct species with a related extant species, the African elephant (Loxodonta africana). An anatomical description of the preserved brain of the woolly mammoth is provided, along with a series of quantitative analyses of various brain structures. These descriptions are based on visual inspection of the actual specimen as well as qualitative and quantitative comparison of computed tomography imaging data obtained for the woolly mammoth in comparison with magnetic resonance imaging data from three African elephant brains. In general, the brain of the woolly mammoth specimen examined, estimated to weigh between 4,230 and 4,340 g, showed the typical shape, size, and gross structures observed in extant elephants. Quantitative comparative analyses of various features of the brain, such as the amygdala, corpus callosum, cerebellum, and gyrnecephalic index, all indicate that the brain of the woolly mammoth specimen examined has many similarities with that of modern African elephants. The analysis provided here indicates that a specific brain type representative of the Elephantidae is likely to be a feature of this mammalian family. In addition, the extensive similarities between the woolly mammoth brain and the African elephant brain indicate that the specializations observed in the extant elephant brain are likely to have been present in the woolly mammoth.

Mass spectrometry compatible surfactant for optimized in-gel protein digestion.

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5-2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20-30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment.

A fossil brain from the Cretaceous of European Russia and avian sensory evolution.

Fossils preserving traces of soft anatomy are rare in the fossil record; even rarer is evidence bearing on the size and shape of sense organs that provide us with insights into mode of life. Here, we describe unique fossil preservation of an avian brain from the Volgograd region of European Russia. The brain of this Melovatka bird is similar in shape and morphology to those of known fossil ornithurines (the lineage that includes living birds), such as the marine diving birds Hesperornis and Enaliornis, but documents a new stage in avian sensory evolution: acute nocturnal vision coupled with well-developed hearing and smell, developed by the Late Cretaceous (ca 90Myr ago). This fossil also provides insights into previous 'bird-like' brain reconstructions for the most basal avian Archaeopteryx--reduction of olfactory lobes (sense of smell) and enlargement of the hindbrain (cerebellum) occurred subsequent to Archaeopteryx in avian evolution, closer to the ornithurine lineage that comprises living birds. The Melovatka bird also suggests that brain enlargement in early avians was not correlated with the evolution of powered flight.

Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1.

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3' ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis.

SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are "nonfilamentous". That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.

PCR-based detection of a rare linear DNA in cell culture.

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10(7) or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.