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Product Quality and Process
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
The most accurate check of protein identity is based on the primary structure, or the protein’s amino acid sequence. This is generally done by the classical Edman degradation using an automated protein sequencer or by liquid chromatography followed by mass spectrometry. In Edman degradation, complex proteins with multiple chains are separated into single chain molecules. Large proteins are then enzymatically digested into fragments of less than 50 amino acids long. Next, the target protein or peptide is immobilized onto a solid and subjected to chemical hydrolysis from the protein’s N-terminus. The released amino acid is made to fluoresce and identified in HPLC. By cycling through the terminal amino acid degradation process, the entire sequence of the protein can be established.
Quality Control of rDNA-Derived Human Tissue-Type Plasminogen Activator
Published in Anthony S. Lubiniecki, Large-Scale Mammalian Cell Culture Technology, 2018
Andrew J. S. Jones, Robert L. Carnick
The most powerful method available for evaluating the fidelity and consistency of the primary structure of proteins the size of rt-PA is peptide mapping. This method has long been used in conjunction with Edman degradation (25) to determine the sequence of amino acids in unknown proteins. Peptide mapping is accomplished by initially reducing and carboxymethylating any disulfide bonds present in the protein. This is followed by cleaving the protein into smaller peptides of usually 25 residues or less by either an enzymatic (typically trypsin) or, occasionally, a chemical method that cleaves the protein at specific sites (see Fig. 5). The resultant peptides are then easily separated by reversed-phase (RP) HPLC, ion-exchange chromatography, two-dimensional thin-layer chromatography (TLC), or gel electrophoresis. A map or “fingerprint” is obtained, which allows for differentiation between proteins of similar, but not identical, primary structure.
Quality and lifecycle management
Published in Sarfaraz K. Niazi, Biosimilars and Interchangeable Biologics, 2016
The primary structure provides information of the amino acid sequence of the protein. For most proteins a primary structure analysis comprises N-terminal sequencing by Edman degradation, C-terminal analysis and peptide mapping followed by HP-RPC purification and subsequent determination of the Mw of the fragment by mass spectroscopy. Peptides and smaller proteins may be sequenced by means of Edman degradation, only. The amino acid sequence is often supported by total amino acid analysis, comparison with the cloned gene sequence and the molecular weight determination. Comparative fingerprints between the natural and the recombinant protein are also used to confirm primary structure identity.
High-level production and purification of bioactive recombinant human activin A in Chinese hamster ovary cells
Published in Preparative Biochemistry & Biotechnology, 2023
Changin Kim, Hyunjoo Kim, Jeong Soo Park, Jiwon Park, Jeongmin Oh, Jaeseung Yoon, Kwanghee Baek
The purity of rhActivin A was assessed during the purification processes using densitometric analysis of SDS gels stained with Coomassie blue. However, the product was visualized using The Molecular Image® Gel Doc™ XR + System (Bio-Rad Laboratories). Additionally, the purity of the finally purified sample was evaluated through size exclusion-high performance liquid chromatography (SE-HPLC) using a Waters® HPLC e2695 Separation Module system (Waters) with a TSK-GEL G3000SWxL column (Tosoh Biosciences). The finally purified rhActivin A (50 µg) was loaded onto a SE-HPLC column with a mobile phase of 300 mM sodium chloride, 100 mM sodium phosphate, pH 6.8 (0.5 mL/min), and UV absorbance was measured at 280 nm. N-terminal amino acid sequencing of the finally purified rhActivin A was performed to verify the identity of the product. Automated Edman degradation using a Procise 492 protein sequencer combined with the HAISIL PTH System (Applied Biosystems) was performed to obtain the N-terminal amino acid sequence. Mass spectrometry of rhActivin A was performed to determine the molecular weight of purified rhActivin A. Native form and reducing form by treatment of 1 M DTT of rhActivin A were prepared and analyzed by LC-ESI-MS. For LC, ZORBAX 300SB-C8 column (Agilent) and Vanquish UHPLC (Thermo Fisher Scientific) were used. For MS, Q Exactive Plus (Thermo Fisher Scientific) was used.
Exploration of green integrated approach for effluent treatment through mass culture and biofuel production from unicellular alga, Acutodesmus obliquus RDS01
Published in International Journal of Phytoremediation, 2019
Silambarasan Tamil Selvan, Balasubramani Govindasamy, Sanjivkumar Muthusamy, Dhandapani Ramamurthy
The native RuBisCO enzyme (which contained amino acid sequence) from A. obliquus was analyzed (under Hewlett Packard G1000A) using the Edman degradation method. The amino acid sequence of rbcL was used for homology search against the SWISS-PROT database using the BLAST program.