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mAB aggregates

Matic Grom, Mirijam Kozorog, Simon Caserman, Andrej Pohar, Blaž Likozar
Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins...
March 1, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Steven A Giannos, Edward R Kraft, Zhen-Yang Zhao, Kevin H Merkley, Jiyang Cai
PURPOSE: Studies were conducted to investigate dilute solutions of the monoclonal antibody (mAb) bevacizumab, mAb fragment ranibizumab and fusion protein aflibercept, develop common procedures for formulation of low concentration mAbs and identify a stabilizing formulation for anti-VEGF mAbs for use in in vitro permeation studies. METHODS: Excipient substitutions were screened. The most stabilizing formulation was chosen. Standard dilutions of bevacizumab, ranibizumab and aflibercept were prepared in PBS, manufacturer's formulation, and the new formulation...
February 28, 2018: Pharmaceutical Research
Peiwu Ding, Shaoshao Zhang, Miao Yu, Yuqian Feng, Qi Long, Huimin Yang, Jingdong Li, Min Wang
Deep venous thrombosis (DVT) is a significant problem in the health care industry worldwide. However, the factors and signaling pathways that trigger DVT formation are still largely unknown. In this study, we investigated the role of interleukin-17A (IL-17A) in DVT formation, focusing on the role of platelet aggregation, neutrophil infiltration, and endothelium cell (EC) activation. Notably, IL-17A levels increased in DVT patients as well as in a mouse DVT model. The DVT model mice were injected with recombinant mouse-IL-17A (rIL-17A) or anti-IL-17A monoclonal antibody (mAb) to further evaluate the effects of this cytokine...
February 23, 2018: International Immunopharmacology
Sarah Hedberg, DongKyu Lee, Yash Mishra, Jonathan Haigh, Daryl R Williams
The osmotic second virial coefficients (B 2 ) which describes protein-protein molecular interactions in solution and were de-termined using self-interaction chromatography (SIC) for an IgG type 1 mAb across a wide range of solution conditions. This data was compared with its time dependent aggregation behaviour as determined using size-exclusion chromatography (SEC) and its temperature dependent aggregation behaviour using dynamic light scattering (DLS) over a 4 week period (SEC) or overnight (DLS). DLS and SEC gave consistent data on aggregation behaviour which correlated well with experimental B 2 trends across the wide pH (4-9) and NaCl concentration (0-1...
February 15, 2018: Analytical Chemistry
Cavan K Kalonia, Frank Heinrich, Joseph E Curtis, Maria Andrea Miller, Steven D Hudson
Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVP). In this work, surface mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters which affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates...
February 9, 2018: Molecular Pharmaceutics
Garrett S Gibbons, Rachel A Banks, Bumjin Kim, Lakshmi Changolkar, Dawn M Riddle, Susan N Leight, David J Irwin, John Q Trojanowski, Virginia M Y Lee
Aggregation of tau into fibrillar structures within the CNS is a pathological hallmark of a clinically heterogeneous set of neurodegenerative diseases termed tauopathies. Unique misfolded conformations of tau, referred to as strains, are hypothesized to underlie the distinct neuroanatomical and cellular distribution of pathological tau aggregates. Here, we report the identification of novel tau monoclonal antibodies (mAbs) that selectively bind to an Alzheimer disease (AD)-specific conformation of pathological tau...
February 5, 2018: Journal of Neuropathology and Experimental Neurology
Rupesh Bommana, Qing Chai, Christian Schöneich, William F Weiss, Ranajoy Majumdar
This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated upon UVA light exposure for up to 72 hours collected by preparative size-exclusion chromatography (SEC), compared to unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by SEC, sodium dodecyl sulfate polyacrylamide gel electrophoresis and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry...
January 30, 2018: Journal of Pharmaceutical Sciences
Yao-Wen Chang, Mika K Kaneko, Shinji Yamada, Yukinari Kato
The mucin-type membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in numerous malignant cancers, including squamous cell carcinoma, germinal neoplasia, mesothelioma, lung cancer, oral cancer, and brain tumor. PDPN expression is strongly associated with cancer progression and poor prognosis. Furthermore, PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation to facilitate tumor metastasis. We have previously reported a novel anti-cat PDPN (cPDPN) monoclonal antibody (mAb), PMab-52, which specifically detects cPDPN using flow cytometry analysis and successfully identifies cPDPN in feline squamous cell carcinomas...
February 2, 2018: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Hisayo Shimizu, Masataka Nakagawa, Nemuri Todaka, Keitaro Imaizumi, Yasunori Kurosawa, Toshiaki Maruyama, C J Okumura, Takashi Shibata, Yosuke Tanaka, Yoshinori Sato, Yasuo Ono, Teruo Akuta
Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods...
January 26, 2018: Protein Expression and Purification
Ehab M Moussa, Satish K Singh, Michael Kimmel, Sandeep Nema, Elizabeth M Topp
Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase...
January 22, 2018: Molecular Pharmaceutics
Richard J Stopforth, Robert J Oldham, Alison L Tutt, Patrick Duriez, H T Claude Chan, Brock F Binkowski, Chad Zimprich, Dun Li, Philip G Hargreaves, Mei Cong, Venkat Reddy, Maria J Leandro, Geraldine Cambridge, Anja Lux, Falk Nimmerjahn, Mark S Cragg
Fc γ receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcγRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain-containing inositol phosphatase 1 to the ITIM of this receptor...
January 19, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
Hyo Helen Chung, Lynette Buck, Kristi Daris, Brent Welborn, Quanzhou Luo, Jette Wypych
Monoclonal antibodies (mAbs) are composed of two heavy chain (HC) and two light chain (LC) polypeptides. The proper folding and assembly of HC and LC is critical for antibody production. Current dogma indicates that the free HCs are retained in the endoplasmic reticulum (ER) unless assembled with LCs into antibodies, while the LCs on the other hand can be secreted as free monomer or dimer molecules. In this study, high levels of extracellular HC homodimers (7%-45%) were observed in the cell culture media during cell line development for mAb1...
January 17, 2018: Biotechnology Progress
Jake W Pawlowski, Adriana Bajardi-Taccioli, Damian Houde, Marina Feschenko, Tyler Carlage, Igor A Kaltashov
Monoclonal antibodies (mAbs) are the fastest growing class of biopharmaceuticals. The specific therapeutic tasks vary among different mAbs, which may include neutralization of soluble targets, activation of cytotoxic pathways, targeted drug delivery, and diagnostic imaging. The specific therapeutic goal defines which interactions of the antibody with its multiple physiological partners are most critical for function, and which ones are irrelevant or indeed detrimental. In this work, we explored the ability of the glycan chains to affect IgG1 interaction with two key receptor families, FcRn and γ-type Fc receptors, as well as the influence of glycan composition on the conformation and stability of the antibody molecule...
January 3, 2018: Journal of Pharmaceutical and Biomedical Analysis
Shinobu Kuwae, Ichiko Miyakawa, Tomohiro Doi
A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 106 cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6...
January 11, 2018: Cytotechnology
Monika Farys, Daniel Gibson, Alan Lewis, Will Lewis, Richard Kucia-Tran
Monoclonal antibodies of the IgG2 and IgG4 isotype were found to exhibit an increased propensity for displaying two-peak elution profiles during cation exchange chromatography. In some cases, this two-peak elution profile also resulted in the formation of non-reversible mAb aggregates. Comparison of IgG1, IgG2 and IgG4 molecules with the same variable region reveals that the two-peak behaviour is predominantly mediated by the constant region and most likely the lower CH1, hinge and upper CH2 regions of the mAb...
January 8, 2018: Biotechnology and Bioengineering
Leon F Willis, Amit Kumar, John Dobson, Nick Bond, David Lowe, Richard Turner, Sheena E Radford, Nikil Kapur, David J Brockwell
Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical sector due to their potency and efficacy against a range of disease targets. These proteinaceous therapeutics are, however, susceptible to unfolding, mis-folding and aggregation by environmental perturbations. Aggregation thus poses an enormous challenge to biopharmaceutical development, production, formulation and storage. Hydrodynamic forces have also been linked to aggregation, but the ability of different flow fields (e.g. shear and extensional flow) to trigger aggregation has remained unclear...
January 8, 2018: Biotechnology and Bioengineering
Yuanli Song, Deqiang Yu, Mukesh Mayani, Nesredin Mussa, Zheng Jian Li
The elucidation of antibody higher order structure (HOS) is critical in therapeutic antibody development. Since HOS determines the protein bioactivity and chemo-physical properties, this knowledge can help to ensure that the safety and efficacy attributes are not compromised. Protein conformational array (PCA) is a novel method for determining the HOS of monoclonal antibodies. Previously, we successfully utilized an enzyme-linked immunosorbent assay (ELISA)-based PCA along with other bioanalytical tools to elucidate the structures of antibody aggregates...
January 9, 2018: MAbs
Johannes S Gach, Margaux Bouzin, Marcus P Wong, Veronika Chromikova, Andrea Gorlani, Kuan-Ting Yu, Brijesh Sharma, Enrico Gratton, Donald N Forthal
Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1...
December 27, 2017: PLoS Pathogens
Albert Jesuran Paul, René Handrick, Sybille Ebert, Friedemann Hesse
Protein aggregation of monoclonal antibodies (mAbs) is a common phenomenon associated with the production of these biopharmaceuticals. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. A preferable approach is to reduce the level of aggregation during bioprocessing by a careful adjustment of critical process parameters. Recently, new analytical methods enabled characterization of mAb aggregation during bioprocessing of mammalian cells...
December 27, 2017: Biotechnology and Bioengineering
June Ereño-Orbea, Taylor Sicard, Hong Cui, Jacob Carson, Pim Hermans, Jean-Philippe Julien
Monoclonal antibodies (mAbs) constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over mAbs for the structural characterization of antibody-antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains...
December 22, 2017: Journal of Molecular Biology
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