Methods for purification, detection and quantification of residual PEI-based transfection reagents

A method for performing an acidic hydrolysis of a liquid mixture comprising a biological matrix and a polyethyleneimine (PEI)-based transfection reagent,

• wherein the biological matrix comprises an adeno-associated virus (AAV) produced using the PEI-based transfection reagent,
• wherein the method comprises incubating the liquid mixture comprising the biological matrix in an aqueous solution comprising 0.1% (v/v) HCl at a temperature of 110° C. for 2 hours,
• wherein said acidic hydrolysis does not degrade the PEI-based transfection reagent, and
• wherein the PEI-based transfection reagent is of general formula (I) or an acceptable salt thereof:
• wherein:
• m represents an integer between 198 to 300 and n represents an integer between 2 to 300, with the proviso that n is lower than m and the sum of min ranges from 200 to 600,
• X represents H,
• p represents an integer between 1 and 4,
• Z represents a group of formula:
• Y0, Y1, Y2, Y3 and Y4, which may be identical or different, represent C or N, with the proviso that at least two, but no more than three, of Y0, Y1, Y2, Y3 and Y4, are N,
• W1, W2, W3 and W4, which may be identical or different, represent H, methyl, cyclopropyl, isopropyl, tert-butyl, phenyl, benzyl, 2-pyridine, 3-pyridine, or 4-hydroxyphenethyl;
• or wherein (i) W1 and W2 or (ii) W2 and W3 or (iii) W3 and W4 together form a fused phenyl;
• a fused phenyl substituted by a methyl group, a methoxy group, a carboxyphenyl or Cl;
• a fused naphthalene;
• a fused 2-pyridine;
• or a fused 3-pyridine,
• with the proviso that at least one, but no more than two, of W1, W2, W3 and W4 is, or are, absent;
• and
• with the proviso that when Y1 and Y2 represent C and Y0, Y3 and Y4 represent N, W1 and W2 do not form a fused 2-pyridine.