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Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography-mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level., Competing Interests: Declaration of competing interest C. Buckley and C. MacHale are employees of Eli Lilly Kinsale Ltd. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Activin receptor type IIA and type IIB fusion protein have been designed to sequester circulating molecules of the transforming growth factor-β (TGF-β) superfamily and inactivate their actions. Members of this superfamily have been reported as essential regulators of erythropoiesis by triggering the formation of activated ternary complexes containing different combinations of type I and type II receptors, which can limit RBC production by accelerating erythroid differentiation and inhibiting erythroid progenitor expansion. The recent approval of Luspatercept for the treatment of anemia associated to transfusion-dependent MDS and Beta-thalassemia in afflicted patients means that it can now pose a real threat of being abused in sport for its ability to stimulate erythropoiesis. Several methods for the detection of these molecules in blood have been proposed for the purpose of sport antidoping control. Here we propose the detection of the ActRIIA-Fc and ActRIIB-Fc fusion proteins by automated capillary Western immunoassay (Simple Western). The use of these immunoassays for the detection of protein targets has become widespread in the recent years. The work presented here demonstrates that this methodology enables a versatile, rapid, and sensitive detection of activin ligand traps in blood samples: plasma, serum, or dried blood spots (DBS). Preliminary results indicate that detection in urine samples is also possible. The option to use different antibodies allows the possibility to use this method as an initial testing procedure as well as a confirmation procedure. Finally, results coming from an administration study confirm that the method is suitable for routine analysis., (© 2022 John Wiley & Sons Ltd.)

Recently, we developed a platform strategy for hinge-deficient human immunoglobulin G1 (IgG1) Fc fusion as a non-immunostimulatory Fc fusion system. As a starting point to establish a promising approach for generating hinge-deficient Fc fusion proteins in Escherichia (E.) coli, we selected a CH2-CH3 scaffold as a model protein for evaluation. Recombinant CH2-CH3, expressed as inclusion bodies, was solubilized with various denaturants (urea, sarkosyl, sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Triton X-100) in neutral (phosphate-buffered saline (PBS), pH 8) or alkaline (50 or 500 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 11) buffer at 25 °C. Similar to the authentic CH2-CH3 produced in Chinese hamster ovary (CHO) cells, all denaturants, except urea in CAPS buffer but not in PBS, were found to elicit the dimer formation of solubilized CH2-CH3 on SDS-polyacrylamide gel electrophoresis (PAGE). After dialysis with PBS, sarkosyl-soluble CH2-CH3 inclusion bodies were successfully purified using protein G-Sepharose, indicating their successful refolding. Compared to the purified CH2-CH3 from its sarkosyl-soluble inclusion bodies in neutral buffer, that in 500 mM CAPS alkaline buffer revealed substantial structure-related similarities, such as secondary structures and thermal stabilities, as measured by circular dichroism spectroscopy, to authentic CH2-CH3. Native PAGE analysis also supported the above data. Therefore, solubilization at alkaline pH is an essential factor that promotes the refolding of CH2-CH3. Dimer formation of CH2-CH3 on SDS-PAGE may act as a surrogate marker for its protein refolding status. Our observations may provide important hints toward downstream processing of Fc-fusion production in E. coli.

Erythropoietin receptor agonists (ERAs) are drugs acting on the early erythropoietic stages developed to treat anemia and other erythropoiesis disease and are prohibited by the World Anti-Doping Agency (WADA). As an alternative to ERAs, a new drug, belonging to the transforming growth factor-b inhibitors family, was recently developed to treat diseases linked to ineffective erythropoiesis. This drug, named as Luspatercept (Reblozyl®), is acting on the later stages of erythropoiesis to promote erythrocytes. This drug might be used by cheating athletes either independently or in combination with ERAs. Indeed, it was shown that Luspatercept and recombinant erythropoietin (rEPO) can act synergistically to increase red blood cells production, potentially allowing the use of lower doses for an efficient effect. Our aim was to find a way to combine the detection of ERAs and Luspatercept without impacting the sensitivity and specificity of ERAs detection from the current techniques implemented in antidoping laboratories and to reduce the time of analysis and total sample volume needed. Magnetic beads coated with antibodies were preferred for IP of samples for its potential multiplexing. Then, the following steps of the method were selected considering that SAR/SDS-PAGE are the electrophoretic methods authorized for initial testing procedure by WADA and that biotinylated primary antibodies used for the immunodetection results in the best sensitivity and specificity and is time saving. The method developed in this work for the combined detection of agents affecting erythropoiesis (AAEs) showed specificity, sensitivity, and robustness and is easily and quickly implementable to all antidoping laboratories., (© 2021 John Wiley & Sons, Ltd.)

Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc-Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of isoelectric points and complex glycosylation profiles including multiple N- and O-linked glycosylation sites. In this review, we highlight the wide range of analytical strategies used to fully characterize Fc-fusion proteins. We also present case studies on the structural assessment of all commercially available Fc-fusion proteins, based on the features and critical quality attributes of their ligand-binding domains., (© 2020 Wiley-VCH GmbH.)

The continuous bed bioreactor systems have been used for the production of protein therapeutics, such as IgG, using immobilized enzyme in biopharmaceutical applications. We developed macroporous poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) cryogel-based bioreactor matrix using sodium dodecyl sulfate as surfactans in the presence of ethylene glycol dimethacrylate as cross linking agent by bulk polymerization. The developed polyGMA immobilized bioreactor with papain enzyme was used for specific fragmentation of immunoglobulin G. The catalysis efficiency for immobilized enzyme were investigated in comparison with free enzyme. The immobilized papain displayed broad catalytic activity over a variety of conditions, with maximal activity around pH 7.0 and 70 °C. The Michaelis-Menten kinetic constant (K m ), the maximum reaction velocity (V max ), and the catalytic efficiency (k cat ) for free enzyme were 0.1097 mg/mL, 29.9 mg/mL/min, and 92.01 1/min, respectively, whereas for immobilized enzyme, K m , V max , and k cat values were 0.1078 mg/mL, 30.53 mg/mL/min, and 94.3 1/min, respectively. In a further step, after digestion, remarkable digestion products of bioreactor, Fab and Fc fragments, produced with immobilized papain bioreactors were analyzed in two ways by SDS-PAGE and reversed-phase HPLC; it was demonstrated that papain immobilized bioreactor successfully used for the digestion of human IgG with high activity. Therefore, the polyGMA cryogel immobilized with papain exhibited a very effective matrix for the bioreactor which can be considered as an alternative bioreactor matrix with great promise in biopharmaceutical applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Monitoring of factor IX (FIX) replacement therapy in haemophilia B relies on accurate coagulation assays. However, considerable interlaboratory variability has been reported for one-stage clotting (OSC) assays. This study aimed to evaluate the real-world, interlaboratory variability of routine FIX activity assays used in clinical haemostasis laboratories for the measurement of recombinant FIX Fc fusion protein (rFIXFc) activity., Methods: Human FIX-depleted plasma was spiked with rFIXFc at 0.80, 0.20 or 0.05 IU/mL based on label potency. Participating laboratories tested samples using their own routine OSC or chromogenic substrate (CS) assay protocols, reagents and FIX plasma standards. Laboratories could perform more than one measurement and method, and were not fully blinded to nominal activity values., Results: A total of 142 laboratories contributed OSC results from 175 sample kits using 11 different activated partial thromboplastin time (aPTT) reagents. The median recovered FIX activity for the 0.80, 0.20 and 0.05 IU/mL samples was 0.72 IU/mL, 0.21 IU/mL and 0.060 IU/mL, respectively. Across all OSC reagents, interlaboratory variability (% CV) per aPTT reagent ranged from 9.4% to 32.1%, 8.2% to 32.6% and 12.2% to 42.0% at the 0.80, 0.20 and 0.05 IU/mL levels, respectively. CS results showed excellent median recoveries at all nominal levels (87.5% to 115.0%; n = 11) with low interlaboratory variability (CV 3.6% to 15.4%)., Conclusion: This large, real-world data set indicates that rFIXFc activity in plasma samples can be accurately measured with the majority of routine OSC and CS assay methods. Given the variation in FIX assay procedures between sites, it is important that individual laboratories qualify their in-house methods for monitoring of rFIXFc activity., (© 2020 Swedish Orphan Biovitrum AB. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Rationale: Glycosylation on immunoglobulins is important for the immune function. In this study, we developed and validated a method for the absolute quantification of IgA subclasses and relative quantification of IgA-Fc glycopeptides by using affinity purification and ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS). Only micro-volumes of plasma were required from each sample and we also applied the method to discover IgA and IgA-glycopeptide profiles in patients with chronic kidney diseases and IgA nephropathy., Methods: Peptide M affinity beads were used to purify IgA, and a cost-effective peptide analogue was added as internal standard. With an efficient on-bead digestion process, purified samples were analyzed by UHPLC/MS/MS in multiple reaction monitoring mode., Results: Correlation coefficients were greater than 0.999 for the IgA1 and IgA2 calibration curves and greater than 0.994 for glycopeptide regression curves. Intraday and interday precisions for IgA1 and IgA2 were <1.6% and <5.1% RSD, respectively. Intraday and interday accuracies ranged from 102.6 to 114.9% and 103.5 to 113.5% for IgA1 and IgA2, respectively. Stabilities of IgA1 and IgA2 at -80°C for 7 to 15 days ranged from 96.0 to 109.4%, respectively. The Pearson's correlation coefficient was 0.916 when comparing the IgA quantification results of the 30 clinical samples by using ELISAs and the developed UHPLC/MS/MS method. Compared with healthy controls, IgA and IgA-glycopeptides showed different profiles in patients with chronic kidney diseases and IgA nephropathy., Conclusions: The developed method showed good validation results, and the absolute quantification results of IgA correlated with those from ELISA. The pilot application study showed that IgA and IgA-glycopeptides can be potential biomarker candidates for kidney diseases, and more clinical sample applications are worth investigating., (© 2019 John Wiley & Sons, Ltd.)

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

Introduction: Variability in FVIII measurement is a recognized problem. There are limited data for samples containing recombinant Factor VIII Fc fusion protein (rFVIIIFc). Many studies use samples for which factor concentrate has been spiked into FVIII deficient plasma in vitro. This approach requires validation., Aim/methods: Four samples were distributed in a UK National External Quality Assessment Scheme for Blood Coagulation (NEQAS BC) survey. One contained Advate (full-length recombinant FVIII) (rFVIII) added to FVIII deficient plasma, one was from a severe haemophilia A patient after infusion of Advate, one was prepared by addition of rFVIIIFc (marketed as Elocta/Eloctate) to FVIII deficient plasma and the fourth was collected from a severe haemophilia A patient following rFVIIIFc (Eloctate) infusion. Fifty-three haemophilia centres (UK and Scandinavia) performed one-stage FVIII assays and 27 performed chromogenic FVIII assays., Results/conclusions: One-stage assays gave significantly lower results than chromogenic assays by 7% (P < 0.01) and 13%(P < 0.001) for post-Advate and Advate spiked samples, and by 22% (P < 0.001) and 23% (P < 0.001) for post-rFVIIIFc and rFVIIIFc spiked samples. The interlaboratory variation was similar for all samples, with CVs of 12%-16% (chromogenic) and 10%-13% (one stage). The data indicate that either product can be safely monitored by one-stage or chromogenic assay. Spiked samples behaved in a similar way to post-infusion samples for both products and could be substituted for post-infusion samples for use in proficiency testing exercises (ie, samples were commutable)., (© 2018 John Wiley & Sons Ltd.)

Unexpected O-glycosylations, including O-xylosylations and mucin-type O-glycosylations, have been reported in recent glycosylation analyses of Fc-fusion proteins produced in mammalian cell expression systems. This observation suggests that therapeutic proteins with novel structures can undergo unintended O-glycosylations, having implications regarding their efficacy and safety. Therefore, the implementation of O-glycosylation analysis during product developmental is essential. However, detail site-specific O-glycosylation analysis is difficult because no consensus sequence for mucin-type O-glycosylations is known, and O-glycopeptides often contain multiple or continuous glycosylation sites. Recently, a new mass spectrometric fragmentation method called electron-transfer/higher-energy collisional dissociation (EThcD) has been used for site-specific glycosylation analysis. In this study, we conducted site-specific O-glycosylation analysis of commercially available GLP1-Fc fusion protein with (G4S) 3 linker peptide using liquid chromatography/mass spectrometry (LC/MS) with EThcD and a glycoproteomic database search. We successfully identified unexpected O-xylosylations at Ser residues in the (G4S) 3 linker peptide, mucin-type O-glycosylations at Thr and Ser residues in the GLP-1 peptide, and Ser residues in the (G4S) 3 linker peptide. This study is the first to report these unexpected O-xylosylations and mucin-type O-glycosylations in this therapeutic fusion protein. Mammalian-cell production of therapeutic fusion proteins that contain novel structures may require exhaustive O-glycosylation analysis to ensure their quality, efficacy, and safety., (Copyright © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Therapeutic proteins are a continuously growing class of pharmaceuticals and comprise several drug candidates with potential performance-enhancing properties. In particular, activin receptor competitors, such as the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc), have the potential for being misused as doping agents in sports as they were found to inhibit negative regulators of late-stage erythropoiesis. Within this study, ammonium sulfate precipitation, immunoaffinity purification, tryptic digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to develop an assay for the combined detection of Sotatercept and Luspatercept in doping control serum samples. The assay was optimized, comprehensively characterized, and found to be fit-for-purpose for application to sports drug testing. It complements existing tests for ActRII-Fc fusion proteins and expands the range of available detection methods for novel protein therapeutics., (© 2018 John Wiley & Sons, Ltd.)

The neonatal Fc receptor (FcRn) binds to the Fc domain of IgG in a pH-dependent manner, guides the intracellular movement of the bound antibodies and protects them from lysosomal degradation. Proper characterization of Fc-FcRn interactions is fundamental to successful design, development, and production of Fc-containing therapeutic proteins because of the potential impact of such interactions on their in vivo pharmacokinetic behaviors. Here, we describe the development and characterization of a cell-based, label-free FcRn-mediated transcytosis assay that provides a functional readout to reflect the totality of Fc-FcRn interactions, including pH-dependent association and dissociation, as well as the intracellular trafficking of Fc-containing molecules in complex with FcRn. Our study demonstrates that this transcytosis assay can be used to evaluate FcRn binding of therapeutic antibodies and Fc-fusion proteins, including wild-type and engineered Fc variants with varying FcRn binding affinities, as well as oxidized and aggregated antibody samples. These results support the utility of an FcRn-dependent transcytosis assay for evaluation of both Fc-FcRn interactions and the structural integrity of Fc-containing therapeutic proteins pertinent to their pharmacokinetic behavior in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have used hydrogen exchange-mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Protein tyrosine sulfation (Tyr-O-SO3) is a common post-translational modification (PTM), which is important for protein function. Absolute quantitation of Tyr-O-SO3 in recombinant therapeutic proteins has been challenging. We report here an MRM method used for absolute quantitation of Tyr-O-SO3 in the hydrolysate of a recombinant Fc-fusion protein. Quantitation is achieved by monitoring the sum of two transitions: the loss of carboxylic acid from tyrosine sulfate (major transition) and sulfate group from tyrosine sulfate sodium salt. The method exhibits a good sensitivity with a limit of quantitation of 1.4 ng/mL, linearity over three orders of magnitude, good repeatability, precision and accuracy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Fc Array is a multiplexed assay that assesses the Fc domain characteristics of antigen-specific antibodies with the potential to evaluate up to 500 antigen specificities simultaneously. Antigen-specific antibodies are captured on antigen-conjugated beads and their functional capacity is probed via an array of Fc-binding proteins including antibody subclassing reagents, Fcγ receptors, complement proteins, and lectins. Here we present the results of the optimization and formal qualification of the Fc Array, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. Assay conditions were optimized for performance and reproducibility, and the final version of the assay was then evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Luspatercept (ACE-536, ACVR2B-Fc), a fusion protein consisting of the extracellular domain of ActRIIB receptor and the Fc-part of human immunoglobulin G1 (IgG1), is currently under clinical development (Phase III). It stimulates the formation of red blood cells and hence may be misused by athletes for doping purposes in the future. Several antibody-based strategies for the detection of Luspatercept and other ACVR2B-Fc fusion proteins in human serum were evaluated (ELISA; IEF-, SDS-, and SAR-PAGE followed by Western blotting; immunoprecipitation). Two methods led to useful results: a commercial "soluble" ACTR-IIB ELISA, which also detected Luspatercept and other ACVR2B-Fc's, but showed no cross-reactivity with Sotatercept/ACVR2A-Fc's. The ELISA might be applied as fast screening tool (100 μL serum; limit of detection (LOD) ca 15.6 ng/mL). The second method uses a polyclonal ACVR2B-antibody for immunoprecipitation followed by SAR-PAGE and Western blotting with a monoclonal detection antibody (50 μL serum; LOD ca 1.0 ng/mL). It can be used for initial as well as for confirmatory testing. Due to the high doses (mg/kg) and long serum half-life of Luspatercept, both strategies may be useful in anti-doping control in the future. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Measurement of recombinant protein product titer critically underpins all biopharmaceutical manufacturing process development, as well as diverse research and discovery activity. Here, we describe a simple rapid (<2 min per 96 samples) 96-well microplate-based assay that enables high-throughput quantitation of recombinant immunoglobulin G and Fc-containing IgG derivatives in mammalian cell culture supernatant over a wide dynamic range of 2.5-80 mg/L, using microplate fluorescence polarization (FP) spectroscopy. The solution-phase FP assay is based on the detection of immunoglobulin Fc domain containing analyte binding to FITC-conjugated recombinant Protein G ligand to measure analyte concentration dependent changes in emitted FP. For ease of use and maximal shelf life, we showed that air-dried assay microplates containing pre-formulated ligand that is re-solubilized on addition of analyte containing solution did not affect assay performance, typically yielding an across plate coefficient of variation of <1%, and a between-plate standard deviation below 1%. Comparative assays of the same samples by FP and other commonly used IgG assay formats operating over a similar dynamic range (Protein A HPLC and bio-interferometry) yielded a coefficient of determination >0.99 in each case., (Copyright © 2017. Published by Elsevier Inc.)

Flexible and protease resistant (G4S) n linkers are used extensively in protein engineering to connect various protein domains. Recently, several groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MS n . The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker [217-221], and first 2 amino acids of a growth factor [222-223]. The linker peptide was first subjected to MS 2 with collision-induced dissociation (CID) activation. The fragmentation profile localized the modification to the GGGPA [218-222] portion of the peptide. Accurate mass measurement indicated that the modification is an addition of an oxygen and cannot be CH 4 , thus eliminating several possibilities such as Pro→Leu. However, other possibilities cannot be ruled out. Higher-energy collision-induced dissociation (HCD)-MS 2 and MS 3 using CID/CID were both unable to differentiate between Ala222→ Ser222 or Pro221→ Hyp221. Finally, MS 3 using high-resolution CID/HCD confirmed the mass increase to be a Pro221→Hyp221 post-translational modification.

Monitoring and evaluation of critical quality attributes (cQA) in monoclonal antibodies (mAb) are a regulatory requirement in pharmaceutical industry. High molecular weight (HMW) species are of critical importance due to the potential risk associated with immunogenicity. HMW species are typically monitored by size exclusion chromatography (SEC). Although low molecular weight (LMW) species are also detected by SEC, low-resolution separation of LMW limits its capability to monitor mAb fragmentation. Recently, we have developed new methods for LMW characterization and evaluation based on the Wes instrument from ProteinSimple. The capillary western blot is based upon size-based separation in a capillary system, and detection by specific immunoprobing, following the separation. The capability of this method for characterization of mAb fragments were demonstrated. The characterization was achieved by probing two antibodies targeted to specific regions (Fc region or Fab region) of IgG1 protein. The specificity of these two antibodies was evaluated against F (ab') 2 and Fc/2 fragments generated from Ides enzyme treated IgG1 protein. The results showed the selected antibodies provide high specificity to F (ab') 2 and Fc/2 fragments. Fractions collected from SEC were used to evaluate this method. The detected fragments from SEC fractions were identified based on their estimated molecular weight and antibody detection. The result proved the capability of the capillary western blot as a characterization method for IgG1 fragments. In addition, with the specific detection to IgG1 and IgG4, the power of the capillary western blot to specifically characterize and evaluate individual IgG fragmentations in an IgG1 and IgG4 mixture was also demonstrated. When heat stressed samples were used, results showed method capability as stability indicating in IgG1 and IgG4 mixture samples. The stressed mixture samples were also evaluated by the total protein assay in which protein samples were biotinylated after separation and were labeled with HRP linked streptavidin to provide chemiluminescence detection. The results indicated total protein assay can be a useful complementary method to capillary western blot immunoassay., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The characterization of complex protein mixtures represents one of the biggest challenge in many research fields such as biological or biopharmaceutical sciences. Out of all categories, monoclonal antibodies (mAbs) and related products drawn the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need for analytical methods to provide comprehensive in-depth characterization of these proteins. In this work, we developed a methodology using CE-UV/MALDI-MS to perform top-down or middle-down characterization after fraction collection enrichment applied to intact protein and mAbs samples. The performance of the method was evaluated with the rapid separation of three intact protein mixture. Good robustness of CZE separation and quality of MALDI-MS spectra and MALDI-ISD spectra of each protein confirms the usefulness of sample enrichment to obtain adequate quantity of deposed protein for top-down analysis and the proof of principle of the method. In a second step, the method was applied to the middle-down characterization of Fc/2 cetuximab variants. Identification of around 9% sequence coverage of Fc/2 cetuximab fragments allows to conclude on the feasibility of the strategy for middle-down characterization of Fc/2 cetuximab variants using CE-UV/MALDI-MS. Moreover, MALDI-ISD fragmentation of Fc/2 cetuximab variants confirm separation phenomenon based on the formation of Fc/2 dimers with and without C-terminal truncation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products.

Therapeutic mAbs play an important role in the treatment of a wide range of diseases. Due to their complexity, comprehensive evaluation of their pharmacokinetics has yet to be fully achieved. It is crucial to develop sensitive, accurate, reliable, and reproducible methods for quantitation of mAbs in complex samples. In addition, it is also important to evaluate the PTMs which can affect their safety and/or effectiveness. MS-based methods provide an emerging approach for quantitation of proteins and their modification forms. In this review, we give a brief overview of quantification analysis of mAbs in complex biological samples and the characterization of N-glycosylation by biological MS., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human therapeutic immunoglobulin gamma (IgG) molecules contain an N-glycan on each of their Fc CH2 domains. These glycans include high-mannose, hybrid, and complex types. Recombinant IgG molecules containing high-mannose glycans have been shown to clear faster in human blood, and exhibit decreased thermal stability. The molecular mechanism behind these observations, however, is not well understood. In this work, we used hydrogen/deuterium exchange combined with mass spectrometry (HDX MS), as well as proteolytic degradation under a native-like condition, to assess the impact of different glycoforms on the molecular structure and stability of recombinant IgG1 and IgG2 molecules expressed from Chinese hamster ovary cells. Our HDX MS data indicate that the conformation of these IgG molecules was indeed influenced by the glycan structure. IgG molecules containing high-mannose and hybrid glycans showed more conformational flexibility in the CH2 domain. This conclusion was further supported by the analysis of glycopeptides released from these molecules by trypsin digestion under a native-like condition. The higher CH2 conformational flexibility of IgG molecules with high-mannose and hybrid glycans contributes to their decreased thermal stability. IgG molecules containing sialylated glycans in the CH2 domain exhibited similar enzymatic degradation behavior as high-mannose glycans, suggesting decreased CH2-domain stability compared to shorter complex glycans, likely resulting from steric effect that decreased the glycan-CH2 domain interaction.

Biosimilarity assessments are performed to decide whether 2 preparations of complex biomolecules can be considered "highly similar." In this work, a machine learning approach is demonstrated as a mathematical tool for such assessments using a variety of analytical data sets. As proof-of-principle, physical stability data sets from 8 samples, 4 well-defined immunoglobulin G1-Fragment crystallizable glycoforms in 2 different formulations, were examined (see More et al., companion article in this issue). The data sets included triplicate measurements from 3 analytical methods across different pH and temperature conditions (2066 data features). Established machine learning techniques were used to determine whether the data sets contain sufficient discriminative power in this application. The support vector machine classifier identified the 8 distinct samples with high accuracy. For these data sets, there exists a minimum threshold in terms of information quality and volume to grant enough discriminative power. Generally, data from multiple analytical techniques, multiple pH conditions, and at least 200 representative features were required to achieve the highest discriminative accuracy. In addition to classification accuracy tests, various methods such as sample space visualization, similarity analysis based on Euclidean distance, and feature ranking by mutual information scores are demonstrated to display their effectiveness as modeling tools for biosimilarity assessments., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated high-mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, size exclusion HPLC, and capillary isoelectric focusing confirmed that the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor, FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods using biolayer interferometry, 1 with protein G-immobilized IgG1 Fc and the other with streptavidin-immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The high-mannose IgG1 Fc and Man5-IgG1 Fc glycoforms were highly similar to one another with high affinity for FcγRIIIa, whereas GlcNAc-Fc had weak affinity, and the nonglycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These 4 IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles and then used as a model system to mathematically assess overall biosimilarity, as described in a series of companion articles., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Amidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein., Methods: To make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway., Results: PAM was equally active when targeted to each intracellular location and assayed in homogenates. Immunocytochemical analyses of CHO cells and a pituitary cell line demonstrated that targeting of exogenous PAM was partially successful. PAM substrates generated by expressing peptidylglycine substrates (glucagon-like peptide 1-Gly, peptide YY-Gly and neuromedin U-Gly) fused to the C-terminus of immunoglobulin Fc in CHO cell lines producing targeted PAM. The extent of amidation of the Fc-peptides was determined by mass spectrometry and amidation-specific enzyme immunoassays. Amidation was inhibited by copper chelation, but was not enhanced by the addition of additional copper or ascorbate., Conclusions: Peptide amidation was increased over endogenous levels by exogenous PAM, and targeting PAM to the endoplasmic reticulum or trans-Golgi network increased peptide amidation compared to endogenous CHO PAM.

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

Immunoconjugates that deliver cytotoxic payloads to cancer cells represent a promising class of therapeutic agents which are intensively investigated in various clinical applications. Prerequisites for the generation of effective immunoconjugates are antibodies which efficiently deliver the respective cytotoxic payload. To facilitate the selection of human or mouse antibodies that display favorable characteristics as immunotoxins, we developed a novel Pseudomonas exotoxin A (ETA)-based screening protein. The α-Fc-ETA' consists of a multispecies-specific Fc-binding domain antibody genetically fused to a truncated ETA version (ETA'). α-Fc-ETA' non-covalently bound to human and mouse antibodies but did not form immune complexes with bovine immunoglobulins. In combination with antibodies harboring human or mouse Fc domains α-Fc-ETA' inhibited proliferation of antigen-expressing tumor cells. The cytotoxic effects were strictly antibody dependent and were observed with low α-Fc-ETA' concentrations. Mouse antibodies directed against CD7 and CD317/HM1.24 that previously had been used for the generation of functional recombinant immunotoxins, also showed activity in combination with α-Fc-ETA' by inhibiting growth of antigen-positive myeloma and leukemia cell lines. In contrast, α-kappa-ETA', a similarly designed human kappa light chain-specific fusion protein, was only specifically active in combination with antibodies containing a human kappa light chain. Thus, the novel α-Fc-ETA' fusion protein is broadly applicable in screening antibodies and Fc-containing antibody derivatives from different species to select for candidates with favorable characteristics for immunotoxin development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

In an effort to reduce the accumulation of ammonia in culture medium, three different TCA cycle intermediates, (alpha-ketoglutarate (α-KG), citric acid and succinic acid) along with glutamic acid for a comparison, were examined as a substitute for glutamine with rCHO cell line producing a Fc-fusion glycoprotein. Among them, α-KG showed the best production performance. When cells were cultivated with 4 mM α-KG, the final ammonia concentration did not exceed 3 mM, which is less than one fourth of that with 4 mM glutamine. The replacement of glutamine increased the lag phase and reduced cell growth. However, it increased the specific productivity by 2.7-fold, resulting in a 1.3-fold increase in the maximum product concentration. Furthermore, the sialic acid content of the Fc-fusion protein with 4 mM α-KG was higher than that with 4 mM glutamine in all cultures, most likely due to the lower ammonia concentration. The results of Western blotting and activity assays of intracellular α-2,3-sialyltransferase and extracellular sialidases are in good agreement with tests done to assess the sialic acid content of the Fc-fusion protein. Taken together, the data obtained here demonstrate that α-KG is a potential substitute for glutamine for improved glycoprotein production in rCHO cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is crucial for antibody effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. To monitor IgG Fc glycosylation, high-throughput techniques for glycosylation analysis are needed in the biotechnology industry. Here we describe the development of a fully automated high-throughput method based on glycopeptide analysis. Samples are prepared in 96-well plates. The IgG's are purified directly from fermentation broths by means of immobilized protein A followed by trypsin digestion. Glycopeptides are purified by hydrophilic interaction solid-phase extraction and analyzed by electrospray mass spectrometry in the positive-ion mode. Data are automatically processed and relative intensities of the various IgG glycopeptides are obtained. The intermediate precision of the method is below 5% for the five major glycoforms of an IgG1 antibody. The newly developed method is suitable for glycosylation profiling of IgG's from fermentation broths. We compared the developed method to other glycoanalytical methods and successfully applied it to analyze the fermentation time course of two different clones of the same therapeutic antibody., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Effective bioanalytical support for pharmacokinetic assessment of therapeutics in early development remains challenging. Concurrent evaluation of multiple candidates per program is typical, requiring efficient characterization in various preclinical species; an ambitious effort often complicated by assay reagent unavailability and limited sample volume. Accordingly, a universal anti-human Fc assay for human monoclonal antibody and derived therapeutics was developed using a microfluidics platform to address these bioanalytical challenges., Results: The universal assay with standardized format was qualified for quantitation of human IgG Fc-containing biotherapeutics in matrices from commonly used preclinical species. Results from this assay compared well with those from traditional colorimetric immunoassays. Furthermore, result comparison between the universal and target-specific assays provided additional information on the effect of antidrug antibodies and in vivo drug catabolism., Conclusion: This assay has wide applicability as a default bioanalytical approach in therapeutic candidate selection and preliminary pharmacokinetics evaluation during early-stage therapeutic development.

IgG functions rely on interactions of the Fc region with other proteins, which are optimized by tailoring of a conserved N-linked glycosylation at Asn-297. We conducted a study involving 735 control individuals and 138 thyroid cancer patients. Here we demonstrated that previously described age-related change in Fc-glycosylation was further characterized by definite sex specificity. In females, the incidences of most of glycosylated forms began to pose characteristic changes at ages of puberty or menopause. In addition, glycan-glycan relationships existed extensively within Fc glycosylation, which were characterized to be altered upon different states of subjects, such as age, sex and thyroid cancer. In thyroid cancer patients, detailed comparison of glycosylation incidences with control individuals yielded insight into aberrant change in IgG(1) Fc-glycosylation. This aberrant pattern was also featured by remarkable specificities of both age and sex. The receiver operating characteristic curve analysis was used to determine diagnostic values of Fc glycosylation. Finally, clinical measurement of two major female sex hormones estradiol and progesterone was conducted to determine potential associations of hormones with IgG Fc glycosylation. This study provided an important view to the associations of IgG Fc N-linked glycosylation with age, sex, female sex hormones and thyroid cancer. This article is part of a Special Issue entitled: Proteomics: The clinical link., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This unit includes protocols for the analysis of the targeting properties of Fc chimeric proteins and antibodies in mice in vivo. We present procedures to prepare the reagents to be tested under endotoxin-free conditions, suitable injection sites, methodology for tissue collection, and processing, and labeling procedures to detect the injected material in situ., (© 2012 by John Wiley & Sons, Inc.)

A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling.

The N-glycan at Asn297 of the immunoglobulin G Fc fragment modulates cellular responses of the adaptive immune system. However, the underlying mechanism remains undefined, as existing structural data suggest the glycan does not directly engage cell surface receptors. Here we characterize the dynamics of the glycan termini using solution NMR spectroscopy. Contrary to previous conclusions based on X-ray crystallography and limited NMR data, our spin relaxation studies indicate that the termini of both glycan branches are highly dynamic and experience considerable motion in addition to tumbling of the Fc molecule. Relaxation dispersion and temperature-dependent chemical shift perturbations demonstrate exchange of the α1-6Man-linked branch between a protein-bound and a previously unobserved unbound state, suggesting the glycan samples conformational states that can be accessed by glycan-modifying enzymes and possibly glycan recognition domains. These findings suggest a role for Fc-glycan dynamics in Fc-receptor interactions and enzymatic glycan remodeling.

The biological activity of therapeutic proteins is strongly dependent on the stability of their folded state, which can easily be compromised by degradation. Oxidation is one of the most common causes of degradation and is typically associated with impairment of the native protein structure. Methionine residues stand out as particularly susceptible to oxidation by reactive oxygen intermediates even under mild conditions. Consequently, methionine oxidation has profound effects on protein activity up to the point of adverse biological responses. Of immediate importance therefore is finding affordable approaches for rapid detection of methionine oxidation before any substantial structural changes can ensue. Herein we report that vibrational bands at 1,044 and 1,113 cm⁻¹ in the mid-infrared region can serve as characteristic markers of methionine oxidation in oxidatively stressed protein therapeutics, monoclonal antibodies (IgG1 and its antigen-binding fragment). Such Fourier-transform infrared (FTIR) markers underpin rapid detection assays and hold particular promise for correlation of methionine oxidation with protein structure and function.

Subcutaneous administration of human IgG is an alternative to intravenous replacement therapy that is associated with more stable serum IgG levels and fewer systemic adverse events. Highly concentrated IgG solutions are most convenient to minimize infusion volume, but their preparation and stability presents substantial technical difficulties. We report on the stability and local tolerance of IgPro20, an l-proline-stabilized, 20% polyvalent human IgG developed for subcutaneous administration. Stability was tested according to ICH guidelines. Local tolerance and vasoactivity were examined in rabbit and rat models, respectively. The presence of l-proline in IgPro20 reduced viscosity and addition of Polysorbate 80 and inert gassing improved the appearance of the solution. After storage at 25 °C for 24 months, monomer + dimer content, aggregates, and fragments were within specification (≥ 90.0%, ≤ 4.0%, and ≤ 10.0%, respectively), and Fc function and antibody activities were maintained. In rats, intravenous injection of IgPro20 produced mild and transient hypotension comparable to that seen with intravenous IgG products. Local tolerance of IgPro20 in rabbits was comparable to that of a marketed subcutaneous IgG, Beriglobin P. Functionality and quality of IgPro20 are maintained during storage at 25 °C for at least 24 months. The product is well tolerated as assessed in animal models., (Copyright © 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

In the efforts to explore an aptamer-based approach for target sensing and detection with higher sensitivity and specificity, instead of directly labeling aptamer with fluorophores, we proposed a new strategy by attaching a polymerase chain reaction (PCR) template to an oligonucleotide aptamer selected by systematic evolution of ligands by exponential enrichment (SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative PCR (RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals. Using the subtractive SELEX method, the oligonucleotide aptamers specific for the Fc fragment of mouse IgG were selected and subjected to coupling with the PCR dsDNA template by using overlap and the asymmetric extension PCR method. The target binding affinity of the PCR template tethered aptamer has been proven by electrophoretic mobility shift assay (EMSA), and further template tethered aptamer mediated real-time quantitative PCR (A-PCR) was conducted to validate the application for such a template tethered aptamer to be a sensitive probe for IgG detection. The results show that the protocols of A-PCR can detect 10-fold serial dilutions of the target, demonstrating a new mechanism to convert aptamer target binding events to amplified RT-PCR signal, and the feasibility of the PCR template tethered aptamer as a facile, specific, and sensitive target probing and detection is established. This new approach also has potential applications in multiple parallel target detection and analysis in a wide range of research fields.

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met[O]) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.

A well-designed anti-drug antibody (ADA) immunoassay is critical for appropriately monitoring the immunogenicity profile of a therapeutic protein during its development. AMG 386 is a peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of angiopoietins with the Tie2 receptor. In bridging immunoassays for ADA, interference by the drug target, present in the assay sample, can result in false positive antibody detection. We used a statistical design-of-experiments approach to identify angiopoietin interference in bridging immunoassays of anti-AMG 386 antibodies. We also demonstrated that a high-affinity monoclonal antibody, directed against an epitope on angiopoietin that competes with AMG 386 binding, could inhibit the angiopoietin interference while preserving the detection of ADA. This report describes the development and validation of methodologies for evaluating and addressing drug target interference in bioanalytical assays that involve interactions between drug, ADA, immune complexes, and drug target., (Copyright 2010 Elsevier B.V. All rights reserved.)

In biopharmaceutical process development, it is desirable to identify sites of covalent degradations to ensure product consistency. One characterization method used for therapeutic immunoglobulin gamma (IgG) 1 antibodies is limited LysC proteolysis followed by reversed-phase LC/MS. Limited LysC proteolysis leads to high efficiency cleavage at the C-terminal side of the hinge lysine 222 residue, generating Fab and Fc fragments. In this report, we show that IgG 1 samples incubated under mildly acidic conditions at elevated temperatures were partially resistant to LysC cleavage at the hinge and resulted in a species where one of the Fab arms remained connected to the Fc region (Fab-Fc). The growth of the Fab-Fc species was proportional to the duration and storage temperature of the incubation period and correlated with the amount of isomerization of the aspartic acid residue preceding lysine 222, determined by peptide mapping. The isomerization rates of samples stored for up to one year at 4 degrees C, 6 months at 29 or 37 degrees C, or 3 months at 45 degrees C were determined, and the activation energy for this conversion was calculated to be approximately 33 kJ mol(-1). The apparent isomerization rate constant was only 0.02 week(-1) for samples stored at 4 degrees C, which resulted in a modest increase from 5.1 to 6.0% isoD after twenty four weeks of storage and, hence, is not a significant concern under normal storage conditions typically used for monoclonal antibodies. However, when stored at 29 degrees C, the apparent rate constant of this reaction was found to be 0.06 week(-1) and resulted in an increase from 5.1 to 21.1% isoD after twenty four weeks of storage and is a major degradant in stressed IgG 1 antibodies.

Background and Objectives: The Fc function assay of immunoglobulin G is performed to ensure that the biological activity of the molecule is not compromised during purification and storage. The current British Pharmacopoeia (BP) method is cumbersome, time consuming and requires a continuous source of fresh red blood cells. We have modified the technique to improve the convenience, throughput and reproducibility of the assay by using frozen red blood cells, a microtitre plate format and electronic derivative analysis for processing results., Materials and Method: Immunoglobulin G samples were assayed using either the BP Fc function procedure or a new procedure incorporating frozen cells, microtitre format and electronic derivative analysis., Results: The Fc function results demonstrated that there was no significant difference between the BP and the new improved procedure., Conclusion: In this study, we demonstrated that a new Fc function assay incorporating frozen red blood cells, microtitre format and derivative analysis of haemolysis curves has been shown to be comparable to the standard BP procedure. It offers a more convenient and quicker means of performing analysis of Fc function of immunoglobulins.

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).

The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 microL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps., ((c) 2008 Wiley Periodicals, Inc.)

Nitration of a recombinant human monoclonal antibody was carried out in vitro by incubating the antibody with the nitrating reagent tetranitromethane (TNM). The susceptible sites of nitration were identified using high-performance liquid chromatography/mass spectrometry (HPLC/MS). In general, tyrosine residues in the variable domains of the antibody are more susceptible to nitration, while tyrosine residues in the constant domains are relatively resistant to nitration. However, one tyrosine residue in the CH1 domain and one tyrosine residue in the CH2 domain are highly susceptible to nitration. Interestingly, the susceptible tyrosine residue in the CH2 domain is followed by the conserved asparagine residue that is glycosylated.

The immunoglobulin degrading enzyme of Streptococcus pyogenes, IdeS, is an unusual cysteine protease produced by group A streptococci for which the only known substrate is immunoglobulin G (IgG). To date, IdeS has not been found to cleave any of the known synthetic substrates that other cysteine proteases hydrolyse, thus making the development of an IdeS detection assay difficult. Furthermore, at high doses of substrate, product generation is inhibited potentially due to the need for a dimeric enzyme complex with IgG. In this study we have developed a mass spectral assay for IdeS activity based on the detection of an Mr approximately 25,300 Fc fragment that retains the ability to bind streptococcal protein G. Using this assay procedure, evidence for a multimeric enzyme-substrate complex was obtained as well as identifying isolated heavy chains as a non-substrate inhibitor of IdeS activity. Under appropriate experimental conditions the assay could be used to detect IdeS activity in bacterial culture media or in human plasma without a requirement for purified reactants. The availability of a rapid and sensitive assay for IdeS should facilitate the detailed biochemical characterization of this unusual bacterial cysteine protease.

Cetuximab is a novel therapeutic monoclonal antibody with two N-glycosylation sites: a conserved site in the CH2 domain and a second site within the framework 3 of the variable portion of the heavy chain. The detailed structures of these oligosaccharides were successfully characterized using orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked oligosaccharides were released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated by normal phase high-performance liquid chromatography (NP-HPLC). The fluorescent-labeled oligosaccharide pool and fractions were analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction was further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of 21 distinct oligosaccharide structures in cetuximab. These oligosaccharides differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation (zero-, mono-, di-, and alpha(1-3)-galactosidase). The individual oligosaccharides were further assigned to the specific sites in the Fab and Fc regions of the antibody. This study represents a unique approach in that MS/MS data were used to identify and confirm the oligosaccharide structures of a protein.