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Vishal Kamat, Ashique Rafique
Rapid growth in the field of biotherapeutics has led to an increased demand for high-throughput, label-free biosensors exhibiting high sensitivity. To support the current needs, Sierra Sensors introduced a surface plasmon resonance imaging (SPRi) based biosensor, Molecular Affinity Screening System (MASS-1). We assessed the potential utility of MASS-1 to support Regeneron's therapeutic antibody discovery. A large panel of antibody-antigen interactions was characterized using MASS-1 and the kinetic data were compared with the Biacore 4000 biosensor...
February 18, 2017: Analytical Biochemistry
Jayant Arora, Sangeeta B Joshi, C Russell Middaugh, David D Weis, David B Volkin
Multi-dose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of four APs, m-cresol, phenol, phenoxyethanol and benzyl alcohol, on conformational stability, aggregation propensity and backbone flexibility of an IgG1 monoclonal antibody, mAb-4, is investigated. Compared to no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by DSC and extrinsic fluorescence spectroscopy...
February 14, 2017: Journal of Pharmaceutical Sciences
Naveen Kumar, Sonal Gupta, Surbhi Dabral, Shailja Singh, Seema Sehrawat
Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death...
February 16, 2017: Molecular and Cellular Biochemistry
Sang Ho Park, Youn Jee Chung, Jae Yen Song, Sang Il Kim, David Pépin, David T MacLaughlin, Patricia K Donahoe, Jang Heub Kim
Müllerian inhibiting substance/anti-Müllerian hormone (MIS/AMH) has been suggested as a biotherapeutic agent in gynecological cancers that highly express the MIS/AMH type II receptors (MISRII/AMHRII) but the anticancer mechanisms by which MIS/AMH acts are not fully understood. Our experiments show that MIS/AMH inhibits ovarian cancer by deregulating the Wnt signal pathway via the β-catenin interacting protein (ICAT). MIS/AMH inhibition of ICAT by small interfering RNAs (siRNA) decreased ICAT driven ovarian cancer cell viability as measured by the methylthiazoltetrazolium assay, reversed cell cycle arrest and annexin V expression and diminished migration by scratch wound assay...
March 2017: International Journal of Oncology
Christian Lanshoeft, Sarah Cianférani, Olivier Heudi
The quantitative analysis of human immunoglobulin G1 (hIgG1) by mass spectrometry is commonly performed using surrogate peptides after enzymatic digestion. Since some limitations are associated with this approach, a novel workflow is presented by hybridizing ligand binding assay (LBA) with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for hIgG1 quantification directly at the intact protein level. Different hIgG1s, including a [(13)C]-labeled version used as internal standard, were immuno-enriched from rat serum with a fully automated platform based on streptavidin coated tips and a biotinylated mouse anti-hIgG capture antibody targeting the fragment crystallizable region followed by overnight deglycosylation prior to LC-HRMS analysis...
February 9, 2017: Analytical Chemistry
Marton Szigeti, Andras Guttman
Temperature gradient capillary electrophoresis was introduced to enhance separation selectivities for branched glycans of biotherapeutic interest. A mixture of afucosylated, fucosylated, and high mannose oligosaccharides was separated in the range of 15 to 45 °C at 5 °C temperature intervals. It was found that within this temperature range, the separation selectivity was carbohydrate structure dependent. The resolution between some glycan structures was greater at elevated temperatures, while others separated better at lower temperatures...
February 3, 2017: Analytical Chemistry
Mara C Inniss, Kalpanie Bandara, Barbara Jusiak, Tim K Lu, Ron Weiss, Liliana Wroblewska, Lin Zhang
As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site-specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system...
February 10, 2017: Biotechnology and Bioengineering
Lorna Ashton, Victoria L Brewster, Elon Correa, Royston Goodacre
In this study we demonstrate the use of Raman spectroscopy to determine protein modifications as a result of glycosylation and iron binding. Most proteins undergo some modifications after translation which can directly affect protein function. Identifying these modifications is particularly important in the production of biotherapeutic agents as they can affect stability, immunogenicity and pharmacokinetics. However, post-translational modifications can often be difficult to detect with regard to the subtle structural changes they induce in proteins...
February 8, 2017: Analyst
Uri Hanania, Tami Ariel, Yoram Tekoah, Liat Fux, Maor Sheva, Yehuda Gubbay, Mara Weiss, Dina Oz, Yaniv Azulay, Albina Turbovski, Yehava Forster, Yoseph Shaaltiel
Plant produced glycoproteins contain N-linked glycans with plant-specific residues of β(1,2)-xylose and core α(1,3)-fucose, which do not exist in mammalian derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy (ERT) does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana...
February 3, 2017: Plant Biotechnology Journal
K Ilker Sen, Robert Hepler, Hirsh Nanda
Methionine oxidation is a prevalent modification found in proteins both in biological settings and in the manufacturing of biotherapeutic molecules. In cells, the oxidation of specific methionine sites can modulate protein function or promote interactions that trigger signaling pathways. In biotherapeutic development, the formation of oxidative species could be detrimental to the efficacy or safety of the drug product. Thus, methionine oxidation is a critical quality attribute that needs to be monitored throughout development...
February 2, 2017: Current Protocols in Protein Science
Maria Monica Castellanos, Arnold McAuley, Joseph E Curtis
In order to increase shelf life and minimize aggregation during storage, many biotherapeutic drugs are formulated and stored as either frozen solutions or lyophilized powders. However, characterizing amorphous solids can be challenging with the commonly available set of biophysical measurements used for proteins in liquid solutions. Therefore, some questions remain regarding the structure of the active pharmaceutical ingredient during freezing and drying of the drug product and the molecular role of excipients...
2017: Computational and Structural Biotechnology Journal
Renpeng Liu, Xuan Chen, Junia Dushime, Megan Bogalhas, Alexandru C Lazar, Thomas Ryll, Lintao Wang
Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis...
January 31, 2017: MAbs
Joël Doré, Marie-Christine Multon, Jehan-Michel Béhier
From the moment of birth, each human being builds a microbe-host symbiosis which is key for the preservation of its health and well-being. This personal symbiotic coexistence is the result of progressive enrichments in microorganism diversity through external supplies. This diversity is nowadays massively overthrown by drastic changes related to clinical practice in birth management, environmental exposure, nutrition and healthcare behaviors. The last two generations have been the frame of massive modifications in life and food habits, with people being more and more sedentary, overfed and permeated with drugs and pollutants...
January 3, 2017: Thérapie
Babasola Sola-Oladokun, Eamonn P Culligan, Roy D Sleator
Bioengineered probiotics represent the next generation of whole cell-mediated biotherapeutics. Advances in synthetic biology, genome engineering, and DNA sequencing and synthesis have enabled scientists to design and develop probiotics with increased stress tolerance and the ability to target specific pathogens and their associated toxins, as well as to mediate targeted delivery of vaccines, drugs, and immunomodulators directly to host cells. Herein, we review the most significant advances in the development of this field...
January 11, 2017: Annual Review of Food Science and Technology
Carlos Eduardo Espinosa-de la Garza, Rodolfo Daniel Salazar-Flores, Néstor Octavio Pérez, Luis Francisco Flores-Ortiz, Emilio Medina-Rivero
Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules...
January 16, 2017: Journal of Visualized Experiments: JoVE
Thomas Beuchert Kallehauge, Shangzhong Li, Lasse Ebdrup Pedersen, Tae Kwang Ha, Daniel Ley, Mikael Rørdam Andersen, Helene Faustrup Kildegaard, Gyun Min Lee, Nathan E Lewis
Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling...
January 16, 2017: Scientific Reports
Jonathan Herskovitz, Josiah Ryman, Theingi Thway, Stephanie Lee, Lei Zhou, Narendra Chirmule, Bernd Meibohm, Vibha Jawa
In the clinical setting, anti-drug antibodies (ADA) against biotherapeutics can influence patient safety and interfere with product efficacy. High immunogenicity has been addressed in clinic by concomitant immune suppression, such as co-administration of methotrexate with enzyme replacement therapy (ERT) and combination tacrolimus/sirolimus treatment for prophylaxis against organ transplant rejection. This study investigates the use of such immune suppressants in mitigating ADA responses to a fully human monoclonal antibody (mAb1) in preclinical animal studies...
January 9, 2017: AAPS Journal
Majors J Badgett, Barry Boyes, Ron Orlando
Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts...
January 3, 2017: Journal of the American Society for Mass Spectrometry
Anica Lancuški, Aiman Abu Ammar, Ron Avrahami, Rita Vilensky, Gleb Vasilyev, Eyal Zussman
Effective encapsulation and protection of biotherapeutics using a bio-based carrier, preferably issued from renewable resources, remains a challenge. Herein, we demonstrate application of coaxial electrospinning to fabric starch-based core-sheath compound fibers as a bacterial cells' carrier. Starch-formate is employed as an encapsulation agent, while the fiber core is made of glycerol, serving as a cell suspension medium. SEM microscopy reveals a distinct core-sheath morphology of the starch-formate/glycerol (SFG) compound fibers with mean diameters of 4...
February 20, 2017: Carbohydrate Polymers
Dandan Li, Cornelus F van Nostrum, Enrico Mastrobattista, Tina Vermonden, Wim E Hennink
Many biomolecules, such as proteins and genes, are presently used as therapeutics. However, their delivery to target sites inside cells is challenging because of their large molecular size, difficulties to pass cellular membranes and their susceptibility for enzymatic and chemical degradation. Nanogels, three-dimensional networks of hydrophilic polymers, are attractive carrier systems for these biotherapeutics because they protect the biologicals against degradation and, importantly, facilitate cell internalization...
December 23, 2016: Journal of Controlled Release: Official Journal of the Controlled Release Society
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