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Commercial Scale Manufacturing of Oligonucleotides Under Good Manufacturing Practices
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Jose E. Gonzalez, Richard G. Einig, Patricia Puma, Timothy P. Noonan, Paul E. Kennedy, Bruce G. Sturgeon, Bing H. Wang, Jin-yan Tang
Early purification schemes relied on a relatively direct scale-up of the reverse phase liquid chromatography (RPLC) methodology used routinely for analysis and preparation of small amounts of oligonucleotides (Beaucage, 1993). RPLC removed the DMT-off capped oligonucleotide failure sequences. The eluted DMT-on oligonucleotide was subsequently detritylated with concentrated acetic acid and precipitated in ethanol. The ammonium salt was converted to a sodium salt by use of ion-exchange on Geigon® cation exchange resins charged with sodium, or by chromatography on an anion exchange support. The sodium form of the oligonucleotide was then concentrated by evaporation, and desalted using gel filtration chromatography. While RPLC purification remains an option for use with certain chemistries, the approach using aqueous buffers (Puma, 1996) is superior for large scale manufacturing due to its scalability, adaptability, and final product purity. We routinely use this aqueous purification process at large scale (Figure 3). It incorporates hydrophobic interaction chromatography (HIC) to remove failure sequences lacking the DMT group. The desired product, still bearing a DMT group, is eluted in low salt buffer or water and subsequently detritylated.
Intracellular Peptide Turnover: Properties and Physiological Significance of the Major Peptide Hydrolases of Brain Cytosol
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
The literature contains reports of a membrane-associated form of c-AAP. This membrane-associated form is clearly not an integral membrane protein. The properties of the membrane associated and soluble forms of the enzyme were compared130. Both had identical molecular weights of 100,000. Both were eluted from a Mono Q® column with the same molarity of NaCl, and both were eluted from a hydrophobic interaction chromatography column at the same position. Both had the same isoelectric point of 5.0, and their peptide maps were similar. N-terminal sequences of the first 18 amino acids were identical. This study convincingly demonstrated the identity of the cytosolic and membrane-associated forms of c-AAP. The authors conclude that the membrane associated form of the enzyme is bound to intracellular membranes with its active site facing the cytosol, precluding its termination of the action of neuronally released enkephalins. They further propose that c-AAP may belong to the class of amphitropic proteins as described by Burn131. Such proteins interact with lipids facilitating their association with membranes.
Biologic Drug Substance and Drug Product Manufacture
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
Ajit S. Narang, Mary E. Krause, Shelly Pizarro, Joon Chong Yee
Hydrophobic interaction chromatography (HIC) separates proteins based on their hydrophobicity. HIC utilizes an immobilized ligand, such as a straight chain alkane, on a hydrophilic carbohydrate matrix, such as cross-linked agarose. Protein with sufficient surface hydrophilicity gets adsorbed on the resin via interaction with the immobilized ligand. Hydrophobicity of the proteins and their interaction with the HIC column is influenced by the polarity of the solvent and salt concentration in solution.
Influence of physiochemical properties on the subcutaneous absorption and bioavailability of monoclonal antibodies
Published in mAbs, 2020
Amita Datta-Mannan, Selina Estwick, Chen Zhou, Hiuwan Choi, Nicole E. Douglass, Derrick R. Witcher, Jirong Lu, Catherine Beidler, Rohn Millican
The global molecule hydrophobicity was determined using a hydrophobic interaction chromatography (HIC)-based method. The data were expressed as a relative hydrophobicity interaction percentage for each of the mAbs to allow for comparisons both within and across the three mAb platforms; larger hydrophobicity interaction percent (HIP) values indicate an increased affinity for the HIC matrix. The Platform 1 molecules show similar and relatively low HIP values; the HIP for mAb 1P and mAb 1RE were 1.3% and 0.7%, respectively. In contrast, both the Platform 2 and 3 molecules showed ~10- to ~100-times higher HIP values than the Platform 1 mAbs (Table 2). The Platform 2 constructs showed similar HIP values for mAb 2P and mAb 2RE of ~16% and ~20%, respectively. Platform 3 mAbs had the widest diversity of HIP, with mAb 3P and 3RE displaying values of ~100% and ~12%, respectively.
An accelerated surface-mediated stress assay of antibody instability for developability studies
Published in mAbs, 2020
Marie R.G. Kopp, Adriana-Michelle Wolf Pérez, Marta Virginia Zucca, Umberto Capasso Palmiero, Brigitte Friedrichsen, Nikolai Lorenzen, Paolo Arosio
We next applied hydrophobic interaction chromatography (HIC) to determine protein hydrophobicity, which has been associated with aggregation,41 viscosity and in vivo clearance.78 The principle of the assay relies on modulating the interactions between mAbs and a hydrophobic stationary phase through the application of a salt gradient. Hydrophobic mAbs show longer retention times compared to less hydrophobic mAbs at increasing salt concentrations.79 We observed that the measured variant hydrophobicities correlate with their aggregation propensities under thermally stressed conditions, measured from the total amount of aggregates determined by SEC (rp = 0.70, rs = 0.70; p < 0.01, and rp = 0.75; p < 0.01, rs = 0.54; p = 0.06 in the first and second replicate runs, respectively). However, when comparing the HNSSA and HIC, the determined correlation was non-significant (rp = 0.42, rs = 0.48; p ≥ 0.1, Figure 5). This poor correlation might arise from the different hydrophobic materials used in both assays, and the fact that the hydrophobic nanoparticles used in the HNSSA carry a residual negative charge, which might play a role in the aggregation of the different variants. Moreover, we have observed that the nanoparticles not only trigger the adsorption of the protein on their surface but are also responsible for further aggregation events, which are not probed for with HIC.60
The role of mass spectrometry in the characterization of biologic protein products
Published in Expert Review of Proteomics, 2018
Deepali Rathore, Anneliese Faustino, John Schiel, Eric Pang, Michael Boyne, Sarah Rogstad
HPLC and UHPLC are also often used as standalone techniques to study structural heterogeneity of intact proteins. For example, strong-cation exchange can be used to resolve charge state variants and to identify charge-related degradation products (e.g. deamidation) [37,38]. Similarly, size exclusion chromatography and hydrophobic interaction chromatography are often used to analyze size- and hydrophobic-variants, respectively [38]. Off-line analysis is especially useful when using off-site mass spectrometers, when analysis of only a portion of chromatogram is required, when direct coupling of these techniques with MS is limited due to mobile phase incompatibility with MS and for multiplexing slower separations to rapid MS analysis. Capillary electrophoresis (CE) is another separation technique used for the characterization of biotechnology products that is rapid, reproducible, and highly sensitive [38,39]. When operating in capillary isoelectric focusing or capillary zone electrophoresis modes, CE can provide information on the charge variants [40]. CE coupled with MS (CE-MS) has been used to separate and identify intact proteins [41], PTMs including signal peptide removal, N-terminal methionine excision, and acetylation [42].