When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid
Anal. Chem., 2015, 87 (15), pp 7529–7534
Publication Date (Web): July 21, 2015
With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions.
Synthesis and characterization of Se-adenosyl-L-selenohomocysteine selenoxide.
Received: 20 Aug 2014
Accepted: 16 Oct 2014
Published online: 24 Nov 2014
Selenium is an essential micronutrient in humans due to the important roles of the selenocysteine-containing selenoproteins. Organoselenium metabolites are generally found to be substrates for the biochemical pathways of their sulfur analogs, and the redox chemistry of selenomethionine and some other metabolites have been previously reported. We now report the first synthesis and characterization of Se-adenosylselenohomocysteine selenoxide (SeAHO) prepared via hydrogen peroxide oxidation of Se-adenosylselenohomocysteine (SeAH). The selenoxide SeAHO, in contrast to its corresponding sulfoxide S-adenosylhomocysteine (SAHO), can form hydrate, has an electrostatic interaction between the α-amino acid moiety and the highly polar selenoxide functional group, and readily oxidizes glutathione (GSH) and cysteine thiols.
Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody.
Anal. Chem., Just Accepted Manuscript
Publication Date (Web): August 19, 2014
Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6, and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2, and led to the formation of so-called "acidic species" that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again remind us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity.
Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization
Article first published online: 14 JUL 2014
The formation of aspartyl succinimide is a common post-translational modification of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV-Vis, liquid chromatography mass spectrometry (LC-MS), and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages are imparted by fluorescence labeling including the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, for example, without optimization, 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions, and related processes relevant to protein pharmaceuticals. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
Discovery and Characterization of a Novel Photo-Oxidative Histidine-Histidine Crosslink in IgG1 Antibody Utilizing 18O-labeling and Mass Spectrometry
Anal. Chem., 2014, 86 (10), pp 4940-4948
A novel photo-oxidative crosslinking between two histidines (His-His) has been discovered and characterized in an IgG1 antibody via the workflow of XChem-Finder -18O labeling and mass spectrometry (Anal Chem 2013, 85, 5900-5908). Its structure was elucidated by peptide mapping with multiple proteases with various specificities (e.g., trypsin, Asp-N, and GluC combined with trypsin or Asp-N) and mass spectrometry with complementary fragmentation modes (e.g., collision-induced dissociation (CID) and electron-transfer dissociation (ETD)). Our data indicated that crosslinking occurred across two identical conserved histidine residues on two separate heavy chains in the hinge region, which is highly flexible and solvent accessible. Based on model studies with short peptides, it has been proposed that singlet oxygen reacts with the histidyl imidazole ring to form an endoperoxide and then converted to the 2-oxo-histidine (2-oxo-His) and His+32 intermediates, the latter is subject to a nucleophilic attack by the unmodified histidine; and finally, elimination of a water molecule leads to the final adduct with a net mass increase of 14 Da. Our findings are consistent with this mechanism. Successful discovery of crosslinked His-His again demonstrates the broad applicability and utility of our XChem-Finder approach in the discovery and elucidation of protein cross-linking, particularly without a priori knowledge of the chemical nature and site of crosslinking.
Decreased glutathione and elevated hair mercury levels are associated with nutritional deficiency-based autism in Oman
Received September 16, 2013.
Accepted January 21, 2014.
Genetic, nutrition, and environmental factors have each been implicated as sources of risk for autism. Oxidative stress, including low plasma levels of the antioxidant glutathione, has been reported by numerous autism studies, which can disrupt methylation-dependent epigenetic regulation of gene expression with neurodevelopmental consequences. We investigated the status of redox and methylation metabolites, as well as the level of protein homocysteinylation and hair mercury levels, in autistic and neurotypical control Omani children, who were previously shown to exhibit significant nutritional deficiencies in serum folate and vitamin B12. The serum level of glutathione in autistic subjects was significantly below control levels, while levels of homocysteine and S-adenosylhomocysteine were elevated, indicative of oxidative stress and decreased methionine synthase activity. Autistic males had lower glutathione and higher homocysteine levels than females, while homocysteinylation of serum proteins was increased in autistic males but not females. Mercury levels were markedly elevated in the hair of autistic subjects vs. control subjects, consistent with the importance of glutathione for its elimination. Thus, autism in Oman is associated with decreased antioxidant resources and decreased methylation capacity, in conjunction with elevated hair levels of mercury.
Arginine Modifications by Methylglyoxal: Discovery in a Recombinant Monoclonal Antibody and Contribution to Acidic Species
Anal. Chem., Just Accepted Manuscript
Publication Date (Web): October 29, 2013
Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors, but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increase of molecular weights of 72 and 54 Daltons, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact of cell culture conditions on the critical quality attributes, as we improve manufacturing processes via the quality by design (QbD) approach.
Discovery of Undefined Protein Crosslinking Chemistry: A Comprehensive Methodology Utilizing 18O-labeling and Mass Spectrometry
Anal. Chem., 2013, 85 (12), pp 5900–5908
Publication Date (Web): May 1, 2013
Characterization of protein crosslinking, particularly without prior knowledge of the chemical nature and site of crosslinking, poses a significant challenge due to their intrinsic structural complexity and the lack of a comprehensive analytical approach. Towards this end, we have developed a generally applicable workflow—XChem-Finder that involves four stages. (1) Detection of crosslinked peptides via 18O-labeling at C-termini. (2) Determination of the putative partial sequences of each crosslinked peptide pair using a fragment ion mass database search against known protein sequences coupled with a de novo sequence tag search. (3) Extension to full sequences based on protease specificity, the unique combination of mass, and other constraints. (4) Deduction of crosslinking chemistry and site. The mass difference between the sum of two putative full-length peptides and the crosslinked peptide provides the formulas (elemental composition analysis) for the functional groups involved in each cross-linking. Combined with sequence restraint from MS/MS data, plausible crosslinking chemistry and site were inferred, and ultimately, confirmed by matching with all data. Applying our approach to a stressed IgG2 antibody, ten cross-linked peptides were discovered and found to be connected via thioether originating from disulfides at locations that had not been previously recognized. Furthermore, once the crosslink chemistry was revealed, a targeted crosslink search yielded four additional crosslinked peptides that all contain the C-terminus of the light chain.
Chapter 287 - Peptidyl-Asp Metalloendopeptidase
Handbook of Proteolytic Enzymes (Third Edition), 2013, Pages 1281�1285
The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 850 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 287 is Peptidyl-Asp Metalloendopeptidase.
An Integrated Proteomic Analysis of Major Isoaspartyl-Containing Proteins in the Urine of Wild Type and Protein L-Isoaspartate O-Methyltransferase-Deficient Mice
Anal. Chem., Just Accepted Manuscript
Publication Date (Web): January 17, 2013. Page: 85, 2423–2430
Copyright � 2013 American Chemical Society
The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and non-enzymatic process, ubiquitous both in vivo and in non-biological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC18.104.22.168) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. Thirty-eight putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.
SUBSTRATE-INDUCED CONTROL OF PRODUCT FORMATION BY PROTEIN ARGININE METHYLTRANSFERASE 1 (PRMT1)
Biochemistry. 2013 Jan 8;52(1):199-209. doi: 10.1021/bi301283t. Epub 2012 Dec 21.
Protein arginine methyltransferases (PRMTs) aid in the regulation of many biological processes. Accurate control of PRMT activity includes recognition of specific arginyl groups within targeted proteins, and also the generation of the correct level of methylation, none of which are fully understood. The predominant PRMT in vivo, PRMT1, has wide substrate specificity and is capable of both mono- and dimethylation, which can induce distinct biological outputs. What regulates the specific methylation pattern of PRMT1 in vivo is unclear. We report that PRMT1 methylates a multisite peptide substrate in a non-stochastic manner, with less C-terminal preference, consistent with the methylation patterns observed in vivo. With a single targeted arginine, PRMT1 catalyzed the dimethylation in a semi-processive manner. The degree of processivity is regulated by substrate sequences. Our results identify a novel substrate-induced mechanism for modulating PRMT1 product specificity. Considering the numerous physiological PRMT1 substrates, as well as the distinct biological outputs of mono- and dimethylation products, such fine-tuned regulation would significantly contribute to the accurate product specificity of PRMT1 in vivo and the proper transmission of biochemical information.