A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection

Oxidation of biopharmaceuticals may affect their bioactivity, serum half-life, and (bio)­chemical stability. The Fc domain of IgG monoclonal antibodies (mAbs) contains two methionine residues which are susceptible to oxidation. Here, we present a middle-down approach employing the cysteine protease IdeS under reducing conditions to obtain three mAb subunits of approximately 25 kDa: Fc/2, Fd′, and LC. These subunits were separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and detected by UV spectroscopy as well as Orbitrap mass spectrometry (MS), as well as MS upon all-ion fragmentation (AIF-MS). We evaluated the feasibility of three strategies for absolute quantification of oxidation in the Fc region of hydrogen peroxide-stressed Rituximab, using a single, commercially available software platform both for data acquisition and evaluation: UV spectroscopy, full-scan MS, and monitoring of product ions obtained by AIF-MS. UV spectroscopy showed the lowest limits of quantification (LOQ) (0.96 ng μL^–1) and featured the lowest relative process standard deviation (<i>V</i>_x0%) of 7.2% compared to MS and AIF-MS with LOQs of 1.24–4.32 ng μL^–1 and relative process standard deviations of 9.0–14%, respectively. Our approach is generic in that it allows monitoring and quantification of oxidation in the Fc regions of fully human and humanized IgG1 mAbs as well as of Fc-fusion proteins. This is exemplified by limits of detection of 1.2%, 1.0%, and 1.2% of oxidation in drug products containing the biopharmaceuticals Rituximab, Adalimumab, and Etanercept, respectively. The presented method is an attractive alternative to conventional time-intensive peptide mapping which is prone to artificial oxidation due to extensive sample preparation

Complete NMR Assignment of Succinimide and Its Detection and Quantification in Peptides and Intact Proteins

Detecting and quantifying post-translational modifications (PTMs) in full-length proteins is a challenge, especially in the case of spontaneously occurring, nonenzymatic PTMs. Such a PTM is the formation of succinimide (Snn) in a protein that occurs spontaneously in prone primary sequences and leads typically to an equilibrium between Snn and its hydrolysis products isoaspartate (isoAsp) and aspartate. In order to detect these modifications in proteins by NMR spectroscopy, chemical shift assignments of reference compounds are required. We used peptide synthesis and 2D NMR spectroscopy to assign all ¹H and ¹³C chemical shifts of Snn and isoAsp and found characteristic chemical shift correlations. To provide chemical shift reference data suitable for comparison with data of denatured proteins, we repeated the assignment in 7 M urea (pH 2.3) and in DMSO. Most characteristic of Snn are the two downfield shifted carbonyl chemical shifts, the chemical shift correlations of Cβ-Hβ of Snn and Cα-Hα of the succeeding residue which are clearly distinct from random coil chemical shift correlations. The characteristic 2D NMR fingerprints of Snn were used to detect and quantify this PTM in the model protein lysozyme, the biotherapeutic filgrastim, and the Fc part of immunoglobulin G1. Mass spectrometry (MS) was applied as an additional independent method. The orthogonality of the NMR and MS techniques allows cross-validation, which is especially important to search for subtle PTMs in proteins. Studying PTMs by NMR spectroscopy is a promising method to analyze proteins and peptides from natural sources, recombinant expression, or chemical synthesis

Analytical Cascades of Enzymes for Sensitive Detection of Structural Variations in Protein Samples

Protein function critically depends on structure. However, current analytical tools to monitor consistent higher-order structure with high sensitivity, as for instance required in the development of biopharmaceuticals, are limited. To complement existing assays, we present the analytical cascade of enzymes (ACE), a method based on enzymatic modifications of target proteins, which serve to exponentially amplify structural differences between them. The method enables conformational and chemical fingerprinting of closely related proteins, allowing for the sensitive detection of heterogeneities in protein preparations with high precision. Using this method, we detect protein variants differing in conformation only, as well as structural changes induced by diverse covalent modifications. Additionally, we employ this method to identify the nature of structural variants. Moreover, the ACE method should help to address the limited reproducibility in fundamental research, which partly relates to sample heterogeneities

Collection of annotated collagenases in <i>Bacillus</i> strains blasted against Q81BJ6 (ColA) as protein query.

<p>Collection of annotated collagenases in <i>Bacillus</i> strains blasted against Q81BJ6 (ColA) as protein query.</p

Cloning, Purification and Characterization of the Collagenase ColA Expressed by <i>Bacillus cereus</i> ATCC 14579

<div><p>Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from <i>Clostridium histolyticum</i> and ColA expressed by <i>Clostridium perfringens</i> are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from <i>Bacillus cereus</i> (<i>B</i>. <i>cereus</i>) has been added to the collection of true collagenases. However, the molecular characteristics of <i>B</i>. <i>cereus</i> ColA are less understood. In this study, we identified ColA as a secreted true collagenase from <i>B</i>. <i>cereus</i> ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). <i>B</i>. <i>cereus</i> ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColA^wt) and mutated to a proteolytically inactive (ColA^E501A) version. Recombinant ColA^wt was tested for gelatinolytic and collagenolytic activities and ColA^E501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColA^wt activity with homologous proteases in additional strains of <i>B</i>. <i>cereus sensu lato</i> (<i>B</i>. <i>cereus s</i>.<i>l</i>.) and related clostridial collagenases revealed that <i>B</i>. <i>cereus</i> ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.</p></div

Gelatinolytic activities expressed by different <i>Bacillus</i> species.

<p>(<b>A</b>) Equal amounts of proteins in lysates of <i>B</i>. <i>subtilis (Bs)</i>, <i>B</i>. <i>megaterium (Bm)</i>, <i>B</i>. <i>thuringiensis (Bt)</i>, <i>B</i>. <i>weihenstephanensis (Bw)</i> and <i>B</i>. <i>cereus</i> ATCC 14579 <i>(Bc)</i> were analyzed for proteolytic activity in gelatin zymography. Protein standard (m) indicated molecular weights of gelatinolytic activities. (<b>B</b>) Efficient disruption of bacteria and equal protein amounts were demonstrated by coomassie-stained SDS PAGEs. Protein standard (m) indicated molecular weights of proteins.</p

Cloning, overexpression and activity of <i>B</i>. <i>cereus</i> ATCC 14579 ColA.

<p>(<b>A</b>) ColA is expressed as a 110.1 kDa protein and consists of a 3.3 kDa signal peptide (aa 1–30), a 7.2 kDa propetide (aa 31–92) and a 99.6 kDa C-terminal part (aa 93–960) of ColA. Expression constructs for N-terminally GST-tagged ColA ΔSP (132.8 kDa) and ColA ΔPP (125.6 kDa) were cloned. Glutamic acid (E) 501 in the active center of ColA was exchanged by an alanine (E501A) to create proteolytically inactive ColA. (<b>B</b>) The expression, enrichment and activity of GST-ColA ΔPP^wt and GST-ColA ΔPP^E501A proteins in IPTG-induced <i>E</i>. <i>coli</i> lysates or purified via GST pull down (PD) experiments were analyzed by SDS-PAGE (left panel) and gelatin zymography (middle panel). To purify ColA ΔPP^wt, transformed <i>E</i>. <i>coli</i> (-) were induced by IPTG to stimulate GST-ColA ΔPP^wt expression. After lysing bacteria, GST-ColA ΔPP^wt was bound to GST sepharose and either eluted by glutathione (PD) as a GST fusion protein (GST-ColA ΔPP^wt) or cleaved and eluted with the PreScission protease (PreSc) to obtain the untagged protease ColA ΔPP^wt.</p

ColA is secreted by <i>B</i>. <i>cereus</i> ATCC 14579.

<p><i>B</i>. <i>cereus</i> ATCC 14579 wildtype (wt) and its isogenic <i>ΔplcR</i> deletion mutant were harvested and disrupted after growing in liquid cultures for indicated time periods. Equal protein amounts of bacterial lysates (upper panel) and equal volumes of supernatants were analyzed by gelatin zymography (upper and middle panel) and Western blotting using a polyclonal antibody directed against ColA ΔPP^E501A (lower panel). Recombinant ColA ΔPP (rColA) was used as control.</p

Collagenolytic activity of ColA.

<p>(<b>A</b>) ColA ΔPP from <i>B</i>. <i>cereus</i> ATCC 14579, its inactive version ColA ΔPP^E501A, ColG from <i>C</i>. <i>histolyticum</i> and the protease domain of ColT (ColT^PD) from <i>C</i>. <i>tetani</i> were tested for their peptidase activity using FALGPA as a substrate. * <i>p</i> = 0.0161 indicates statistical significance (Student´s t-test, paired, one-tailed). (<b>B</b>) In <i>in vitro</i> cleavage assays, the positive control ColG and ColA ΔPP^wt were incubated with tropocollagen type I for the indicated time periods and analyzed by coomassie stained SDS PAGE to analyze their collagenolytic activities. (<b>C</b>) As negative controls, ColT^PD and ColAΔPP^E501A were investigated. (<b>D</b>) As indicated, α–chymotrypsin was incubated with tropocollagen type I as an additional negative control and compared to untreated tropocollagen type I for the indicated time periods and analyzed by coomassie stained SDS PAGE.</p