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1.
《MABS-AUSTIN》2013,5(5):578-585
The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed. 相似文献
2.
Yingda Xu Dongdong Wang Bruce Mason Tony Rossomando Ning Li Dingjiang Liu 《MABS-AUSTIN》2019,11(2):239-264
Increasing attention has been paid to developability assessment with the understanding that thorough evaluation of monoclonal antibody lead candidates at an early stage can avoid delays during late-stage development. The concept of developability is based on the knowledge gained from the successful development of approximately 80 marketed antibody and Fc-fusion protein drug products and from the lessons learned from many failed development programs over the last three decades. Here, we reviewed antibody quality attributes that are critical to development and traditional and state-of-the-art analytical methods to monitor those attributes. Based on our collective experiences, a practical workflow is proposed as a best practice for developability assessment including in silico evaluation, extended characterization and forced degradation using appropriate analytical methods that allow characterization with limited material consumption and fast turnaround time. 相似文献
3.
Hongcheng Liu Christine Nowak Mei Shao Gomathinayagam Ponniah Alyssa Neill 《Biotechnology progress》2016,32(5):1103-1112
Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103–1112, 2016 相似文献
4.
Lobvi E. Matamoros Fernndez Drio E. Kalume Loany Calvo Minerva Fernndez Mallo Antonio Vallin Peter Roepstorff 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,752(2):1505
In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies. 相似文献
5.
Christine Nowak Jason K. Cheung Shara M. Dellatore Amit Katiyar Ram Bhat Joanne Sun 《MABS-AUSTIN》2017,9(8):1217-1230
Forced degradation studies have become integral to the development of recombinant monoclonal antibody therapeutics by serving a variety of objectives from early stage manufacturability evaluation to supporting comparability assessments both pre- and post- marketing approval. This review summarizes the regulatory guidance scattered throughout different documents to highlight the expectations from various agencies such as the Food and Drug Administration and European Medicines Agency. The various purposes for forced degradation studies, commonly used conditions and the major degradation pathways under each condition are also discussed. 相似文献
6.
Tetyana Dashivets Jan Stracke Stefan Dengl Alexander Knaupp Jan Pollmann Johannes Buchner 《MABS-AUSTIN》2016,8(8):1525-1535
Therapeutic antibodies can undergo a variety of chemical modification reactions in vitro. Depending on the site of modification, either antigen binding or Fc-mediated functions can be affected. Oxidation of tryptophan residues is one of the post-translational modifications leading to altered antibody functionality. In this study, we examined the structural and functional properties of a therapeutic antibody construct and 2 affinity matured variants thereof. Two of the 3 antibodies carry an oxidation-prone tryptophan residue in the complementarity-determining region of the VL domain. We demonstrate the differences in the stability and bioactivity of the 3 antibodies, and reveal differential degradation pathways for the antibodies susceptible to oxidation. 相似文献
7.
A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG, including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC–MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC–MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA. 相似文献
8.
A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed. 相似文献
9.
Hongcheng Liu Gomathinayagam Ponniah Hui-Min Zhang Christine Nowak Alyssa Neill Nidia Gonzalez-Lopez Rekha Patel Guilong Cheng Adriana Z Kita Bruce Andrien 《MABS-AUSTIN》2014,6(5):1145-1154
Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules. 相似文献
10.
《MABS-AUSTIN》2013,5(5):1145-1154
Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules. 相似文献
11.
12.
SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity
and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are
routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by
matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the
banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody
lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity
of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were
formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and β-elimination are
the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling
can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing
SDS-PAGE. Breakage of disulfide bonds by β-elimination was evidenced by the detection of dehydroalanine using mass spectrometry. 相似文献
13.
Benjamin Moore 《MABS-AUSTIN》2016,8(2):371-378
Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MSn experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified. 相似文献
14.
Alexandre Ambrogelly Stephen Gozo Amit Katiyar Shara Dellatore Yune Kune Ram Bhat 《MABS-AUSTIN》2018,10(4):513-538
Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle. 相似文献
15.
Noritoshi Kitamoto Tomoichiro Oka Kazuhiko Katayama Tian‐Cheng Li Naokazu Takeda Yoji Kato Tatsuya Miyoshi Tomoyuki Tanaka 《Microbiology and immunology》2012,56(11):760-770
Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1–7], 7 GII, [GII.1–7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus‐like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty‐five hybrid clones producing MAbs were obtained. Twenty‐four MAbs were characterized by ELISA, according to their cross‐reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross‐reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup‐specific; and (iii) those reactive in a genotype‐specific manner. Further analysis of three broadly cross‐reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV. 相似文献
16.
Construction of recombinant monoclonal antibodies from a chicken hybridoma line secreting specific antibody 总被引:2,自引:0,他引:2
Naoto Nakamura Yuri Aoki Hiroyuki Horiuchi Shuichi Furusawa Hachiro I. Yamanaka Tetsuyuki Kitamoto Haruo Matsuda 《Cytotechnology》2000,32(3):191-198
The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scFV) mAb by using the variable heavy(VH) and light (VL)genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrPc from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of VH and VL genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system. 相似文献
17.
To study the immunological features of the hepatitis C virus (HCV) envelope protein (E2 protein), new specific monoclonal antibodies (mAbs) were generated. WKA/H rats were immunized with syngeneic cells infected with a vaccinia virus expressing the E2 protein and with soluble E2 protein obtained from Chinese hamster ovary cells with a plasmid-based expression system. By screening hybridoma cells obtained from spleen cells of the immunized rats, three specific mAbs were obtained. One mAb was reactive to a peptide corresponding to the hypervariable region 1 (HVR1) in E2 protein, while the others reacted to regions outside HVR1. The significance of these antibodies for the diagnosis of HCV infection as well as for analysis of the structure of the HCV E2 protein will be discussed. 相似文献
18.
Ronald Levy Yehudit Bergman Olivera Finn 《In vitro cellular & developmental biology. Plant》1981,17(12):1051-1057
Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined
by monoclonal antibodies: Leu-1 and HLA-DR.
Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington,
D.C., June 7–11, 1981.
This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302.
This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech
Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England
Nuclear Corporation, and Ortho Pharmaceutical Corporation. 相似文献
19.
Generation of recombinant antibodies 总被引:7,自引:0,他引:7
Recombinant antibody technology is opening new perspectives for the development of novel therapeutic and diagnostic agents.
In this review we focus on advances in the generation of both genetically engineered humanized and fully human monoclonal
antibodies. Methods for their production in different expression systems are also discussed. 相似文献