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Characterization of Biosimilar Biologics
Published in Laszlo Endrenyi, Paul Jules Declerck, Shein-Chung Chow, Biosimilar Drug Product Development, 2017
The challenge in generating a biosimilar antibody therapeutic is well illustrated in a report of the analysis of a candidate Herceptin biosimilar (Xie et al., 2010). Values for methionine oxidation, deamidation of asparagine and glutamine were reported; however, a note added in press stated that the values were higher than those obtained by the innovator company. It was therefore posited that additional chemical modifications had been introduced into the protein when reconstituting the innovator Herceptin prior to analysis. This illustrates both the susceptibility of such large molecules to chemical modification in vitro and the sensitivity of techniques available for determining comparability. A gross error resulted from generating an IgG1 constant region sequence different from that of the innovator product (Xie et al., 2010). This suggests that rather than sequencing the innovator product prior to embarking on the project, the sequence of Herceptin had been sourced from a sequence database that has been shown to be in error (Jefferis and Lefranc, 2009).
ROW regulatory guidance
Published in Sarfaraz K. Niazi, Biosimilars and Interchangeable Biologics, 2016
Increased knowledge of the relationship between biochemical, physicochemical, and biological properties of the product and clinical outcomes will facilitate development of an SBP. Because of the heterogeneous nature of proteins (especially those with extensive post-translational modifications, such as glycoproteins), the limitations of some analytical techniques, and the generally unpredictable nature of the clinical consequences of minor differences in protein structural/physicochemical properties, the evaluation of comparability will have to be carried out independently for each product. For example, oxidation of certain methionine residues in one protein may have no impact on clinical activity whereas in another protein it may significantly reduce the intrinsic biological activity or increase immunogenicity. Thus, differences in the levels of methionine oxidation in the RBP and SBP would need to be evaluated and, if present, their clinical relevance would be evaluated and discussed.
Reactions With Disinfectants
Published in Richard A. Larson, Eric J. Weber, Reaction Mechanisms in Environmental Organic Chemistry, 2018
Richard A. Larson, Eric J. Weber
Some amino acids are quite rapidly attacked, especially those containing sulfur such as cystine, cysteine, and methionine (Hoigné and Bader, 1983b: Pryor et al., 1984: Menzel, 1971), and attack on enzymes containing these compounds is probably one of the principal mechanisms by which ozone exerts its antibacterial activity. In addition to the above-mentioned compounds, the aromatic amino acids tryptophan, tyrosine, histidine, and phenylalanine are also susceptible to destruction by ozone, as they are with other electrophilic oxidizing agents (Mudd et al., 1969). As in the case of other organic amines, the rates of reaction are proportional to the amount of unprotonated amine present. Few data are available on the reaction products of ozone with these compounds. Methionine sulfoxide was almost the sole product of methionine oxidation at pH 7.2 in phosphate buffer, and cysteine gave a mixture of cystine and cysteic acid, whereas ammonia was a major product of histidine ozonolysis under these conditions (Mudd et al., 1969). Ozonolysis of tyrosine gave phenol-coupled dimer, o, o’–dityrosine (87), as well as 3,4-dihydroxyphenylalanine (DOPA, 88) and polymeric material. In addition, phenylalanine was partly converted to tyrosine and o, o’–dityrosine by ozone. These products are best explained by HO·-initiated free-radical reactions (Verweij et al., 1982). Anilines reacted with aqueous ozone to form a series of oxidation products (azobenzenes, azoxybenzenes, and benzidines: 89–91) that appeared largely to result from attack of ·OH on either the amine group or the aromatic ring, followed by radical-radical coupling and (in some cases) further oxidation (Chan and Larson, 1992).
Analysis of polysaccharide hydrolases secreted by Aspergillus flavipes FP-500 on corn cobs and wheat bran as complex carbon sources
Published in Preparative Biochemistry & Biotechnology, 2020
Lizzete Ruth Torres-Barajas, María Teresa Alvarez-Zúñiga, Guillermo Mendoza-Hernández, Guillermo Aguilar-Osorio
Protein identification was performed from the MS/MS spectra datasets using the MASCOT search algorithm (Matrix Science, London, UK available at http://www.matrixscience.com). Trypsin was set as the specific digest reagent, the precursor mass tolerance and fragmentation tolerances equaled 0.5 and 0.8 Da, respectively. One missed cleavage site was allowed, and carbamidomethyl-cysteine was set as a fixed variable, while methionine oxidation was set as a variable modification. The protein identification reporting criteria included at least two MS/MS spectrum matched at the 95% level of confidence (Mowse score = 25 in the conditions used in this work) and the presence of a consecutive y and/or b ion series of three or more amino acids. (Mowse score = −10X log10 (p), where p is the likelihood that the identification is a random event).
Proteomic analysis of secretomes from Bacillus sp. AR03: characterization of enzymatic cocktails active on complex carbohydrates for xylooligosaccharides production
Published in Preparative Biochemistry & Biotechnology, 2021
Johan S. Hero, José H. Pisa, Enzo E. Raimondo, M. Alejandra Martínez
Peptides identification was performed through Thermo Scientific™ Proteome Discoverer version 2.1 software, Waltham, MA, USA, using the reference proteome of B. subtilis 168 (UP000001570) as database. The following parameters were considered as search criteria: parent ion tolerance, 10 ppm; fragment ion mass tolerance, 0.05 Da; Miscleavage, 2; Dynamic Modifications, methionine oxidation (M); static modifications, Carbamidomethylation (C). On these bases, the software qualified the detected peptides in three confidence levels: Low, Medium, and High. At least two peptides per protein were necessary to consider the presence of a protein in the sample. For area estimations of each identified protein, we only considered those peptides with a High confidence level.
Insights into the N-terminal Cu(II) and Cu(I) binding sites of the human copper transporter CTR1
Published in Journal of Coordination Chemistry, 2018
Yulia Shenberger, Ortal Marciano, Hugo E. Gottlieb, Sharon Ruthstein
All peptides used in this study were synthesized on a rink amide resin (Applied Biosystems, Foster City, CA). Couplings of standard Fmoc (9-fluorenylmethoxy-carbonyl)-protected amino acids were achieved with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, Dchem) in N,N-dimethylformamide (DMF, Bio-Lab) in combination with N,N-diisopropylethylamine (DIPEA, Bio-Lab) for a 1-h cycle. Fmoc deprotection was achieved with piperidine (Bio-Lab). Side-chain deprotection and peptide cleavage from the resin were achieved by treating the resin-bound peptides with a 5-mL cocktail of 90% trifluoroacetic acid (TFA, Bio-Lab), 5% ethane dithiol (EDT, Alfa Aesar, Haverhill, MA), 2.5% triisopropylsilane (TIS, Alfa Aesar), and 2.5% thioanisole (Alfa Aesar), for 2.5 h under N2. An additional 65 μL of bromotrimethylsilane (TMSBr, Alfa Aesar) were added during the final 30 min to minimize methionine oxidation. The peptides were washed four times with cold diethyl ether, vortexed, and then centrifuged for 5 min at 3500 rpm. After evaporation of TFA under N2, 10 mM DTT (dithiothreitol, Sigma-Aldrich, St. Louis, MO) was added to the peptide, and it was dissolved in high-performance liquid chromatography (HPLC) water. The peptide was then purified using preparative reversed-phase HPLC (Vydac, C18, 5 cm). The mass of the peptide was confirmed either by MALDI-TOF MS-Autoflex III-TOF/TOF mass spectrometer (Bruker, Bremen, Germany) equipped with a 337-nm nitrogen laser, or with ESI (electron spray ionization) mass spectrometry on a quadruple time-of-flight (Q-TOF) low resolution micromass spectrometer (Waters Corp., Milford, MA). Peptide samples were typically mixed with two volumes of premade dihydrobenzoic acid (DHB) matrix solution, deposited onto stainless steel target surfaces, and allowed to dry at room temperature.