RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme.

tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5' ends are removed by an endonuclease called RNase P. All RNase P enzymes characterized so far are composed of an RNA plus one or more proteins, and tRNA 5' end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified the components of human mitochondrial RNase P and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that does not require a trans-acting RNA component for catalysis. Moreover, the mitochondrial enzyme turns out to be an unexpected type of patchwork enzyme, composed of a tRNA methyltransferase, a short-chain dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary origin, possibly representing the enzyme's metallonuclease moiety. Apparently, animal mitochondria lost the seemingly ubiquitous RNA world remnant after reinventing RNase P from preexisting components.

Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins.

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development. Graphical abstract.

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 (Vx0%) 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.

Site-specific characterization and absolute quantification of pegfilgrastim oxidation by top-down high-performance liquid chromatography-mass spectrometry.

The characterization and absolute quantification of protein biopharmaceuticals and their product-related impurities, e.g., oxidation variants, is essential due to their potential impact on biological activity and immunogenicity. Here, we present site assignment and absolute quantification of oxidation variants of pegfilgrastim, a poly(ethylene glycol) modified recombinant human granulocyte-colony stimulating factor. Pegfilgrastim stressed with 1.0% hydrogen peroxide served as a model protein for developing a top-down high-performance liquid chromatography-mass spectrometry (HPLC-MS) platform that allowed direct site assignment of Met122, Met127, and Met138 oxidation within a total analysis time of 30 min. Three different absolute quantification methods, namely, UV absorption spectroscopy, full-scan MS, and all-ion fragmentation (AIF) MS were compared. Additionally, the monitoring of all generated fragment ions or selected sets of fragment ions were evaluated for the AIF method. Linearity of calibration curves from 5.0 to 25 ng µL(-1), 25 to 250 ng µL(-1), and 100 to 1000 ng µL(-1) was confirmed. The AIF method achieved a lower limit of detection of 0.85 ng µL(-1) and a lower limit of quantification of 2.54 ng µL(-1). On the basis of the comparison of relative standard deviations of interday measurements, AIF was concluded to be the method of choice for concentrations up to 50 ng µL(-1), and UV measurements should be carried out above this concentration. Finally, an expired pegfilgrastim batch was analyzed as a a real biopharmaceutical sample to confirm the feasibility of our approach for monitoring low levels of oxidation variants.

A direct-infusion- and HPLC-ESI-Orbitrap-MS approach for the characterization of intact PEGylated proteins.

The characterization of proteins modified with poly(ethylene glycol) (PEG), such as recombinant human granulocyte-colony stimulating factor (PEGylated rhG-CSF or pegfilgrastim), by electrospray ionization-mass spectrometry (ESI-MS) constitutes a challenge due to the overlapping protein charge state pattern and PEG polydispersity. In order to minimize spectral overlaps, charge reduction by means of the addition of amine was applied. Method development for direct-infusion measurements, carried out on an ESI-time-of-flight (ESI-TOF) instrument, demonstrated the potential of triethylamine (TEA) for shifting the charge state pattern toward lower-charged species and of formic acid (FA) for causing higher charging. After successful method transfer to the LTQ Orbitrap XL instrument, isotopically resolved mass spectra could be acquired. With a median mass accuracy of 1.26 ppm, a number-average monoisotopic molecular mass of 40074.64 Da was determined for pegfilgrastim. The direct comparison of three Orbitrap mass spectrometers, namely the LTQ Orbitrap XL, the Exactive, and the Q Exactive, demonstrated that online interfacing to high performance liquid chromatography (HPLC) was only feasible with the Q Exactive, which offers adequate spectral quality on a time scale compatible with chromatographic separation (i.e., 0.2 min acquisition time per chromatographic peak). Finally, the applicability of both direct-infusion Orbitrap MS and HPLC interfaced to Orbitrap MS was demonstrated for the detection of methionine oxidation in pegfilgrastim. Singly, doubly, and triply oxidized species were readily resolved in the chromatogram, while their oxidation status was easily determined from the mass shifts observed in the deconvoluted mass spectra.

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase--extensive moonlighting in mitochondrial tRNA biogenesis.

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5' extensions, is the methyltransferase responsible for m(1)G9 and m(1)A9 formation. The ability of the mitochondrial tRNA:m(1)R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5' end processing.

Top-down MS for rapid methionine oxidation site assignment in filgrastim.

Protein therapeutics have emerged as a major new class of pharmaceuticals. One important shelf-life-limiting factor of biopharmaceuticals is methionine oxidation, and therefore, it is important that analytical methods are able to thoroughly characterize all possible oxidized variants. Here, we present a fast and sensitive method to perform online methionine oxidation site assignment using granulocyte colony-stimulating factor (filgrastim) as a model. The method is based on top-down MS using the all-ion fragmentation mode of the Exactive benchtop mass spectrometer. Conditions that provide information on the intact mass of the protein as well as on fragment ions that allow unambiguous site assignment of methionine oxidation in filgrastim variants as low as 0.12% of total peak area in a chromatographic time scale were identified. Using this method, we performed methionine oxidation site assignment in H2O2-stressed filgrastim and in filgrastim which was stored at intended conditions, respectively. We show that the relative abundance of oxidation species observed in filgrastim stored under intended conditions differs strikingly from the oxidized species observed after H2O2 stress. Additionally, we report an oxidized filgrastim variant that has not been previously described in the literature.

Lesson from the stoichiometry determination of the cohesin complex: a short protease mediated elution increases the recovery from cross-linked antibody-conjugated beads.

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analyses-such as the stoichiometry determination of protein complexes-have become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periods-as performed in standard on-bead digestion-led to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with a 1:1:1:1 stoichiometry. Furthermore, using isoform specific reference peptides, we determined the exact STAG1:STAG2 stoichiometry within the population of cohesin complexes. In summary, we show that the protease elution protocol increases the recovery from affinity beads and is compatible with quantitative measurements such as the stoichiometry determination of protein complexes.

Improved precision of iTRAQ and TMT quantification by an axial extraction field in an Orbitrap HCD cell.

Improving analytical precision is a major goal in quantitative differential proteomics as high precision ensures low numbers of outliers, a source of false positives with regard to quantification. In addition, higher precision increases statistical power, i.e., the probability to detect significant differences. With chemical labeling using isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT) reagents, quantification is based on the extraction of reporter ions from tandem mass spectrometry (MS/MS) spectra. We compared the performance of two versions of the LTQ Orbitrap higher energy collisional dissociation (HCD) cell with and without an axial electric field with regard to reporter ion quantification. The HCD cell with the axial electric field was designed to push fragment ions into the C-trap and this version is mounted in current Orbitrap XL ETD and Orbitrap Velos instruments. Our goal was to evaluate whether the purported improvement in ion transmission had a measurable impact on the precision of MS/MS based quantification using peptide labeling with isobaric tags. We show that the axial electric field led to an increased percentage of HCD spectra in which the complete set of reporter ions was detected and, even more important, to a reduction in overall variance, i.e., improved analytical precision of the acquired data. Notably, adequate precision of HCD-based quantification was maintained even for low precursor ion intensities of a complex biological sample. These findings may help researchers in their design of quantitative proteomics studies using isobaric tags and establish HCD-based quantification on the LTQ Orbitrap as a highly precise approach in quantitative proteomics.

Proteomic analysis of endosomes from genetically modified p14/MP1 mouse embryonic fibroblasts.

The p14/MP1 scaffold complex binds MEK1 and ERK1/2 on late endosomes, thus regulating the strength, duration and intracellular location of MAPK signaling. By organelle proteomics we have compared the protein composition of endosomes purified from genetically modified p14⁻/⁻, p14+/⁻ and p14(rev) mouse embryonic fibroblasts. The latter ones were reconstituted retrovirally from p14⁻/⁻ mouse embryonic fibroblasts by reexpression of pEGFP-p14 at equimolar ratios with its physiological binding partner MP1, as shown here by absolute quantification of MP1 and p14 proteins on endosomes by quantitative MS using the Equimolarity through Equalizer Peptide strategy. A combination of subcellular fractionation, 2-D DIGE and MALDI-TOF/TOF MS revealed 31 proteins differentially regulated in p14⁻/⁻ organelles, which were rescued by reexpression of pEGFP-p14 in p14⁻/⁻ endosomes. Regulated proteins are known to be involved in actin remodeling, endosomal signal transduction and trafficking. Identified proteins and their in silico interaction networks suggested that endosomal signaling might regulate such major cellular functions such as proliferation, differentiation, migration and survival.

Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap.

Peptide labeling with isobaric tags has become a popular technique in quantitative shotgun proteomics. Using two different samples viz. a protein mixture and HeLa extracts, we show that three commercially available isobaric tags differ with regard to peptide identification rates: The number of identified proteins and peptides was largest with iTRAQ 4-plex, followed by TMT 6-plex, and smallest with iTRAQ 8-plex. In all experiments, we employed a previously described method where two scans were acquired for each precursor on an LTQ Orbitrap: A CID scan under standard settings for identification, and a HCD scan for quantification. The observed differences in identification rates were similar when data was searched with either Mascot or Sequest. We consider these findings to be the result of a combination of several factors, most notably prominent ions in CID spectra as a consequence of loss of fragments of the label tag from precursor ions. These fragment ions cannot be explained by current search engines and were observed to have a negative impact on peptide scores.

Stoichiometry determination of the MP1-p14 complex using a novel and cost-efficient method to produce an equimolar mixture of standard peptides.

Determination of protein complex stoichiometry can be achieved by absolute quantification of the interacting constituents based on isotope dilution mass spectrometry. Current available platforms for the generation of standard peptides are cost-intensive and deliver variable results concerning the equimolarity of the standard peptides. Here we describe a novel and cost-efficient method to generate an equimolar mixture of standard peptides, which we call the equimolarity through equalizer peptide (EtEP) strategy. The rationale of the strategy allows equalization of internal standard peptides and absolute quantification of any protein of interest via a single peptide, from which the exact amount was determined by amino acid analysis. This and the use of the mTRAQ reagent significantly decrease the costs of absolute quantification and stoichiometry determination. We used the EtEP strategy to determine the MP1-p14 complex stoichiometry of two different concentrations, one simulating a condition following tandem affinity purification. Absolute quantification of MP1-p14 was performed on two different mass spectrometers, and the 1:1 stoichiometry was confirmed with high accuracy and precision. On the basis of the quantification of MP1-p14, we demonstrate the importance to assess completeness of protein digestion and discuss the use of peptides containing labile amino acids and the choice of instrumentation.

Dilute-and-shoot analysis of therapeutic monoclonal antibody variants in fermentation broth: a method capability study.

Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient therapeutic agents for various medical conditions, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drugs. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N-glycosylation and truncation variants of the expressed mAb are identified at the intact protein level. Simultaneously, absolute quantification of mAb content in fermentation broth is achieved. The whole workflow features excellent robustness as well as retention time and peak area stability. Additional enzymatic removal of N-glycans enables determination of mAb glycation levels, which are subsequently considered in relative N-glycoform quantification to correct for isobaric galactosylation. Several molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production, as well as targeted biosimilar development.

Absolute quantification of cohesin, CTCF and their regulators in human cells.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.

Maintaining consistent quality and clinical performance of biopharmaceuticals.

INTRODUCTION: Biopharmaceuticals are large protein based drugs which are heterogeneous by nature due to post translational modifications resulting from cellular production, processing and storage. Changes in the abundance of different variants over time are inherent to biopharmaceuticals due to their sensitivity to subtle process differences and the necessity for regular manufacturing changes. Product variability must thus be carefully controlled to ensure that it does not result in changes in safety or efficacy. AREAS COVERED: The focus of this manuscript is to provide improved understanding of the science and strategies used to maintain the quality and clinical performance of biopharmaceuticals, including biosimilars, throughout their lifecycle. This review summarizes rare historical instances where clinically relevant changes have occurred, defined here as clinical drift, and discusses modern tools used to prevent such changes, including improved analytics, quality systems and regulatory frameworks. EXPERT OPINION: Despite their size complexity and heterogeneity, modern analytics, manufacturing quality systems and comparability requirements for the evaluation of manufacturing changes cumulatively help to ensure the consistent quality and clinical performance of biopharmaceuticals throughout their product lifecycle. Physicians and patients can expect the same safety and efficacy from biopharmaceuticals and their respective biosimilars irrespective of batch or production history.

Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals.

Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom-up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.

The structure-function relationship of disulfide bonds in etanercept.

Etanercept is a TNFα receptor Fc fusion protein used for the treatment of rheumatic disease and psoriasis. Physicochemical and functional investigation of process fractions during development of the etanercept biosimilar GP2015 (Erelzi®) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain. This novel structure-function relationship was found to be the molecular basis for reduced potency in recent Enbrel® batches, which exhibit higher levels of incorrect disulfide bridging. Interestingly, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring potency to normal levels. This redox dependent reversibility suggests that these variants are likely not relevant for clinical efficacy once the drug enters the bloodstream. Nonetheless, incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should thus be carefully monitored and controlled to guarantee patient safety.

Identification of Low-Level Product-Related Variants in Filgrastim Products Presently Available in Highly Regulated Markets.

BACKGROUND: Filgrastim is a recombinant, non-glycosylated form of human granulocyte colony-stimulating factor, used to stimulate leukocyte proliferation in patients suffering from neutropenia. Since the expiration of patents associated with Amgen's filgrastim biopharmaceutical, Neupogen(®), in 2006, a number of filgrastim products have been marketed; however, a detailed characterization and comparison of variants associated with these products have not been publically reported. OBJECTIVE: The objective of this study was to identify and quantify product-related variants in filgrastim reference products and biosimilars thereof that are presently available in highly regulated markets. METHODS: In this study, we used intact and top-down mass spectrometry to identify and quantify product-related variants in filgrastim products. Mass spectrometry has become the method of choice for physicochemical characterization of biopharmaceuticals, allowing accurate and sensitive characterization of product-related variants. RESULTS: In addition to modifications ubiquitously present in biopharmaceuticals, such as methionine oxidation and asparagine/glutamine deamidation, we identified six different low-level, product-related variants present in some, but not all, of the tested products. Two variants, an acetylated filgrastim variant and a filgrastim variant containing an additional C-terminal tryptophan extension, are newly identified variants. CONCLUSION: This study demonstrates that filgrastim products already in widespread clinical use in highly regulated markets differ in low-level, product-related variants present at levels mostly below 1 % relative abundance. This study provides a comprehensive catalog of minor differences between filgrastim products and suggests that the filgrastim product-related variants described here are not clinically relevant when present at low abundance.

Comparability of biosimilar filgrastim with originator filgrastim: protein characterization, pharmacodynamics, and pharmacokinetics.

BACKGROUND: Biosimilars provide safety, purity, and potency similar to those of a reference biologic product. METHODS: An array of protein analytical techniques was used to compare the physicochemical properties of proposed biosimilar filgrastim (EP2006), US-approved originator filgrastim, and EU-approved originator filgrastim. Biological characterization involved surface plasmon resonance spectroscopy analyses and in vitro proliferation assays. A randomized, double-blind, two-way crossover, phase I study in healthy volunteers assessed the pharmacodynamics, pharmacokinetics, and safety profiles of EP2006 and US-approved originator filgrastim (administered as a single subcutaneous 10 µg/kg injection). RESULTS: EP2006 and originator filgrastim (US and EU approved) were highly similar with respect to primary, secondary, and tertiary protein structures; mass, size, purity, charge, and hydrophobicity. No differences in receptor binding affinity were observed, and all samples demonstrated similar in vitro bioactivity. In the phase I study, no statistically significant differences between EP2006 and US-approved originator filgrastim were noted in pharmacodynamic or pharmacokinetic parameters, and all confidence intervals were within the equivalence boundaries. The two products had similar safety profiles. CONCLUSION: These studies provide robust evidence of the structural and functional similarity between the proposed biosimilar filgrastim (EP2006) and the US-approved originator filgrastim.