PEG-antibody

Altogether, the PEGylation approaches presented herein have been successful to small proteins that, overall, have a limited number of active sites on the surface. Such approaches might not be applicable to all proteins, particularly for the larger ones. It is too early to speculate whether those limitations might be responsible for the low anti-PEG antibody response evoked following administration in humans of PEG-uricase [73]. 

Xu, Y., Mehl, IT, Bakhtiar, R., et al. (2010) Immunoaffi-nity purification using anti-PEG antibody followed by two-dimensional liquid chromatography/tandem mass spectrometry tor the quantification of a PEGylated therapeutic peptide in human plasma. Analytical Chemistry, 82, 6877-6886. 

Myler H, Hruska MW, Srinivasan S, Cooney E, Kong G, Dodge R, et al. Anti-PEG antibody bioanalysis a chnical case study with PEG-IFN-lambda-la and PEG-IFN-alpba2a in naive patients. Bioanalysis 2015 7 1093-106. 

Yoshimoto K, Nishio M, Sugasawa H, Nagasaki Y (2010) Direct observation of adsorption-induced inactivation of antibody fragments surrounded by mixed-PEG layer on a gold surface. J Am Chem Soc 132 7982-7989... 

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

Although NHS-LC-biotin and sulfo-NHS-LC-biotin are very popular reagents for biotinylation, they both result in hydrophobic aliphatic biotin modifications on proteins and antibodies. Unfortunately, these groups have a tendency to aggregate in aqueous solution and may cause protein precipitation or loss of activity over time. For this reason, the use of more hydrophilic PEG-based biotin compounds of approximately the same spacer length may be a better alternative for maintaining water solubility of modified proteins (Chapter 18). 

In addition, the PEG-based heterobifunctional crosslinkers described in Chapter 18, Section 2, provide enhanced water-solubility for antibody conjugation applications. Conjugation of antibody molecules using a maleimide-PEG -NHS ester compound actually increases the solubility of the antibody and may help to maintain stability for certain sensitive monoclonals better than the traditional aliphatic crosslinkers. The methods described below for SMCC may be used with success for PEG-based reagents or other maleimide-NHS ester heterobifunctionals. 

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