Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry.


:During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.






Scott RA,Rogers R,Balland A,Brady LJ




Has Abstract


2014-01-01 00:00:00














  • A novel IgE-neutralizing antibody for the treatment of severe uncontrolled asthma.

    abstract::The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought...


    pub_type: 杂志文章


    authors: Cohen ES,Dobson CL,Käck H,Wang B,Sims DA,Lloyd CO,England E,Rees DG,Guo H,Karagiannis SN,O'Brien S,Persdotter S,Ekdahl H,Butler R,Keyes F,Oakley S,Carlsson M,Briend E,Wilkinson T,Anderson IK,Monk PD,von Wachenfe

    更新日期:2014-05-01 00:00:00

  • Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis.

    abstract::Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ...


    pub_type: 杂志文章


    authors: Cao M,De Mel N,Jiao Y,Howard J,Parthemore C,Korman S,Thompson C,Wendeler M,Liu D

    更新日期:2019-08-01 00:00:00

  • Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor.

    abstract::The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients exp...


    pub_type: 杂志文章


    authors: Koefoed K,Steinaa L,Søderberg JN,Kjær I,Jacobsen HJ,Meijer PJ,Haurum JS,Jensen A,Kragh M,Andersen PS,Pedersen MW

    更新日期:2011-11-01 00:00:00

  • Differential effects of IL-2 and IL-21 on expansion of the CD4+ CD25+ Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia.

    abstract::CD4(+) CD25(+) regulatory T cells are expanded in solid and hematological malignancies including chronic lymphocytic leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the ...


    pub_type: 杂志文章


    authors: Gowda A,Ramanunni A,Cheney C,Rozewski D,Kindsvogel W,Lehman A,Jarjoura D,Caligiuri M,Byrd JC,Muthusamy N

    更新日期:2010-01-01 00:00:00

  • Phase 1 study of anti-epidermal growth factor receptor monoclonal antibody in patients with solid tumors.

    abstract::In the present study, we conducted a Phase 1 study of a recombinant anti-EGFR monoclonal antibody (CMAB009) that has the same amino acid sequence as cetuximab. The purpose of this study was to evaluate the safety, pharmacokinetics, and potential benefit of CMAB009 in Chinese patients with advanced chemotherapy-resista...


    pub_type: 杂志文章,随机对照试验


    authors: Wang C,He X,Zhou B,Li J,Li B,Qian W,Hou S,Wang H,Shi Y,Guo Y

    更新日期:2011-01-01 00:00:00

  • Effects of the FcRn developmental pharmacology on the pharmacokinetics of therapeutic monoclonal IgG antibody in pediatric subjects using minimal physiologically-based pharmacokinetic modelling.

    abstract::The aim of this study was to investigate neonatal Fc receptor (FcRn) concentration developmental pharmacology in adult and pediatric subjects using minimal physiologically-based pharmacokinetic (mPBPK) modelling. Three types of pharmacokinetic (PK) data for three agents (endogenous/exogenous native IgG, bevacizumab an...


    pub_type: 杂志文章


    authors: Hardiansyah D,Ng CM

    更新日期:2018-10-01 00:00:00

  • Investigation of monoclonal antibody dimers in a final formulated drug by separation techniques coupled to native mass spectrometry.

    abstract::Therapeutic monoclonal antibodies (mAbs) are highly complex proteins that must be exhaustively characterized according to the regulatory authorities' recommendations. MAbs display micro-heterogeneity mainly due to their post-translational modifications, but also to their susceptibility to chemical and physical degrada...


    pub_type: 杂志文章


    authors: Rouby G,Tran NT,Leblanc Y,Taverna M,Bihoreau N

    更新日期:2020-01-01 00:00:00

  • Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia.

    abstract::The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off‑target effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and...


    pub_type: 杂志文章


    authors: Fitting J,Blume T,Ten Haaf A,Blau W,Gattenlöhner S,Tur MK,Barth S

    更新日期:2015-01-01 00:00:00

  • Certolizumab pegol.

    abstract::Certolizumab pegol (Cimzia(®)) is currently the only PEGylated anti-TNFα biologic approved for the treatment of rheumatoid arthritis and Crohn disease. The product, developed by UCB, is a humanized antigen-binding fragment (Fab') of a monoclonal antibody that has been conjugated to polyethylene glycol. Certolizumab pe...


    pub_type: 杂志文章,评审


    authors: Goel N,Stephens S

    更新日期:2010-03-01 00:00:00

  • Immunotoxins constructed with chimeric, short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity.

    abstract::An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the "deglycosylated" A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appear...


    pub_type: 杂志文章


    authors: Liu XY,Pop LM,Schindler J,Vitetta ES

    更新日期:2012-01-01 00:00:00

  • Ultrafast and high-throughput N-glycan analysis for monoclonal antibodies.

    abstract::Glycosylation is a critical attribute for development and manufacturing of therapeutic monoclonal antibodies (mAbs) in the pharmaceutical industry. Conventional antibody glycan analysis is usually achieved by the 2-aminobenzamide (2-AB) hydrophilic interaction liquid chromatography (HILIC) method following the release...


    pub_type: 杂志文章


    authors: Yang X,Kim SM,Ruzanski R,Chen Y,Moses S,Ling WL,Li X,Wang SC,Li H,Ambrogelly A,Richardson D,Shameem M

    更新日期:2016-05-01 00:00:00

  • A human monoclonal antibody against small envelope protein of hepatitis B virus with potent neutralization effect.

    abstract::Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients ...


    pub_type: 杂志文章


    authors: Wang W,Sun L,Li T,Ma Y,Li J,Liu Y,Li M,Wang L,Li C,Xie Y,Wen Y,Liang M,Chen L,Tong S

    更新日期:2016-01-01 00:00:00

  • Fluorescent Citrine-IgG fusion proteins produced in mammalian cells.

    abstract::Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology, proteomics, microscopy, cell biology and molecular diagnostics, although efficient production of fluorescent IgGs in mammalian cells has been hampered by different and mutually incompatible secretion- and folding-requireme...


    pub_type: 杂志文章


    authors: Haas AK,von Schwerin C,Matscheko D,Brinkmann U

    更新日期:2010-11-01 00:00:00

  • Developability studies before initiation of process development: improving manufacturability of monoclonal antibodies.

    abstract::Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other t...


    pub_type: 杂志文章


    authors: Yang X,Xu W,Dukleska S,Benchaar S,Mengisen S,Antochshuk V,Cheung J,Mann L,Babadjanova Z,Rowand J,Gunawan R,McCampbell A,Beaumont M,Meininger D,Richardson D,Ambrogelly A

    更新日期:2013-09-01 00:00:00

  • Expression of antibodies using single open reading frame (sORF) vector design: Demonstration of manufacturing feasibility.

    abstract::Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chain...


    pub_type: 杂志文章


    authors: Gion WR,Davis-Taber RA,Regier DA,Fung E,Medina L,Santora LC,Bose S,Ivanov AV,Perilli-Palmer BA,Chumsae CM,Matuck JG,Kunes YZ,Carson GR

    更新日期:2013-07-01 00:00:00

  • Improved monovalent TNF receptor 1-selective inhibitor with novel heterodimerizing Fc.

    abstract::The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory r...


    pub_type: 杂志文章


    authors: Richter F,Seifert O,Herrmann A,Pfizenmaier K,Kontermann RE

    更新日期:2019-05-01 00:00:00

  • Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life.

    abstract::The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants...


    pub_type: 杂志文章


    authors: Mackness BC,Jaworski JA,Boudanova E,Park A,Valente D,Mauriac C,Pasquier O,Schmidt T,Kabiri M,Kandira A,Radošević K,Qiu H

    更新日期:2019-10-01 00:00:00

  • An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification.

    abstract::Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is pos...


    pub_type: 杂志文章


    authors: Sotelo P,Collazo N,Zuñiga R,Gutiérrez-González M,Catalán D,Ribeiro CH,Aguillón JC,Molina MC

    更新日期:2012-07-01 00:00:00

  • Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.

    abstract::We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv...


    pub_type: 杂志文章


    authors: Yin G,Garces ED,Yang J,Zhang J,Tran C,Steiner AR,Roos C,Bajad S,Hudak S,Penta K,Zawada J,Pollitt S,Murray CJ

    更新日期:2012-03-01 00:00:00

  • Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies.

    abstract::Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs)...


    pub_type: 杂志文章


    authors: Avery LB,Wang M,Kavosi MS,Joyce A,Kurz JC,Fan YY,Dowty ME,Zhang M,Zhang Y,Cheng A,Hua F,Jones HM,Neubert H,Polzer RJ,O'Hara DM

    更新日期:2016-08-01 00:00:00

  • CD40- and CD95-specific antibody single chain-Baff fusion proteins display BaffR-, TACI- and BCMA-restricted agonism.

    abstract::Antibodies that target a clinically relevant group of receptors within the tumor necrosis factor receptor superfamily (TNFRSF), including CD40 and CD95 (Fas/Apo-1), also require binding to Fc gamma receptors (FcγRs) to elicit a strong agonistic activity. This FcγR dependency largely relies on the mere cellular anchori...


    pub_type: 杂志文章


    authors: Nelke J,Medler J,Weisenberger D,Beilhack A,Wajant H

    更新日期:2020-01-01 00:00:00

  • Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit.

    abstract::Blockade of immune checkpoint pathways by programmed cell death protein 1 (PD-1) antibodies has demonstrated broad clinical efficacy against a variety of malignancies. Sintilimab, a highly selective, fully human monoclonal antibody (mAb), blocks the interaction of PD-1 and its ligands and has demonstrated clinical ben...


    pub_type: 杂志文章


    authors: Wang J,Fei K,Jing H,Wu Z,Wu W,Zhou S,Ni H,Chen B,Xiong Y,Liu Y,Peng B,Yu D,Jiang H,Liu J

    更新日期:2019-11-01 00:00:00

  • American Association of Pharmaceutical Scientists National Biotechnology Conference Short Course: Translational Challenges in Developing Antibody-Drug Conjugates: May 24, 2012, San Diego, CA.

    abstract::The American Association of Pharmaceutical Scientists (AAPS) National Biotechnology Conference Short Course "Translational Challenges in Developing Antibody-Drug Conjugates (ADCs)," held May 24, 2012 in San Diego, CA, was organized by members of the Pharmacokinetics, Pharmacodynamics and Drug Metabolism section of AAP...




    authors: Thudium K,Bilic S,Leipold D,Mallet W,Kaur S,Meibohm B,Erickson H,Tibbitts J,Zhao H,Gupta M

    更新日期:2013-01-01 00:00:00

  • Biosimilars in oncology and inflammatory diseases: current and future considerations for clinicians in Latin America.

    abstract::Biological therapies have revolutionized the treatment of several cancers and systemic immune-mediated inflammatory conditions. Expiry of patents protecting a number of biologics has provided the opportunity to commercialize highly similar versions, known as biosimilars. Biosimilars are approved by regulatory agencies...


    pub_type: 杂志文章,评审


    authors: Scheinberg M,Pineda C,Castañeda-Hernández G,Zarbá JJ,Damião A,Arantes LH Jr,Jacobs I

    更新日期:2018-08-01 00:00:00

  • Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines.

    abstract::The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. The choice of expression system for prod...


    pub_type: 杂志文章


    authors: Yoo EM,Yu LJ,Wims LA,Goldberg D,Morrison SL

    更新日期:2010-05-01 00:00:00

  • High throughput detection of antibody self-interaction by bio-layer interferometry.

    abstract::Self-interaction of an antibody may lead to aggregation, low solubility or high viscosity. Rapid identification of highly developable leads remains challenging, even though progress has been made with the introduction of techniques such as self-interaction chromatography (SIC) and cross-interaction chromatography (CIC...


    pub_type: 杂志文章


    authors: Sun T,Reid F,Liu Y,Cao Y,Estep P,Nauman C,Xu Y

    更新日期:2013-11-01 00:00:00

  • Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries.

    abstract::Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundred...


    pub_type: 杂志文章


    authors: Bhatta P,Whale KD,Sawtell AK,Thompson CL,Rapecki SE,Cook DA,Twomey BM,Mennecozzi M,Starkie LE,Barry EMC,Peters SJ,Kamal AM,Finney HM

    更新日期:2021-01-01 00:00:00

  • Galactosylation variations in marketed therapeutic antibodies.

    abstract::There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary...


    pub_type: 杂志文章


    authors: Raju TS,Jordan RE

    更新日期:2012-05-01 00:00:00

  • The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen.

    abstract::A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-Ig™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the ...


    pub_type: 杂志文章


    authors: Correia I,Sung J,Burton R,Jakob CG,Carragher B,Ghayur T,Radziejewski C

    更新日期:2013-05-01 00:00:00

  • Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces.

    abstract::One aspiration for the formulation of human monoclonal antibodies (mAb) is to reach high solution concentrations without compromising stability. Protein surface activity leading to instability is well known, but our understanding of mAb adsorption to the solid-liquid interface in relevant pH and surfactant conditions ...


    pub_type: 杂志文章


    authors: Couston RG,Skoda MW,Uddin S,van der Walle CF

    更新日期:2013-01-01 00:00:00