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Reversed-phase (RP) HPLC typically uses octadecyl silica (C18) columns in combination with acidic mobile phases, composed of acetonitrile/water [32, 41,42,43], with additives like formic acid (FA) [41, 43, 45, 46], ammonium formate [42, 45, 47, 48], acetic acid (AcA) [49], ammonium acetate [32, 50], trichloroacetic acid (TCA) [38, 51], trifluoroacetic acid (TFA) [32, 50, 52], or ammonium phosphate [53]

Therapeutic proteins would be released in the interstitial space [14] or intracellularly [20] by specific protease sites engineered between the GST and the therapeutic protein

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