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Antibody-Drug Conjugates: Design, Formulation and Physicochemical Stability

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The convergence of advanced understanding of biology with chemistry has led to a resurgence in the development of antibody-drug conjugates (ADCs), especially with two recent product approvals. Design and development of ADCs requires the synergistic combination of the monoclonal antibody, the linker and the payload. Advances in antibody science has enabled identification and generation of high affinity, highly selective, humanized or human antibodies for a given target. Novel linker technologies have been synthesized and highly potent cytotoxic drug payloads have been created. As the first generation of ADCs utilizing lysine and cysteine chemistries moves through the clinic and into commercialization, second generation ADCs involving site specific conjugation technologies are being evaluated and tested. The latter aim to be better characterized and controlled, with wider therapeutic indices as well as improved pharmacokinetic-pharmacodynamic (PK-PD) profiles. ADCs offer some interesting physicochemical properties, due to conjugation itself, and to the (often) hydrophobic payloads that must be considered during their CMC development. New analytical methodologies are required for the ADCs, supplementing those used for the antibody itself. Regulatory filings will be a combination of small molecule and biologics. The regulators have put forth some broad principles but this landscape is still evolving.

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4-(4′-acetylphenoxy) butanoic acid


3-acetylphenyl acetic acid


Anti-drug antibody


Antibody-drug conjugate


Antibody-dependent cell-mediated cytotoxicity


Auristatin F


Area under the curve


Bromoacetamido moiety


Biologics License Application




N-acetyl gamma calicheamicin


Circular dichroism spectroscopy


Complement-dependent cytotoxicity


Chinese hamster ovary cells


Drug antibody ratio


Disulfide-containing maytansinoids


Dimethyl acid linker


Dimethyl hydrazide linker precursor




Drug product


Drug substance


Differential scanning calorimetry


Drug substance intermediate






Enzyme-linked immunosorbent assay


Electrospray ionization mass spectroscopy


Ethylvinyl acetate


Fragment antigen-binding


Neonatal Fc receptor for IgG


Fc gamma receptor for IgG


Fourier transform infrared spectroscopy






Human antibody-human antibody immune response


Heavy chain of IgG


High density polyethylene


Hydrophobic interaction chromatography


Hydrazine-iso-Pictet-Spengler ligation


Human Fc gamma receptor for IgG


Investigational New Drug




Light chain of IgG


Low density polyethylene


Monoclonal antibody


Matrix Assisted Laser Desorption/Ionization Time of Flight


Maleimidocaproyl linker




Monomethyl auristatin D


Monomethyl auristatin E


Monomethyl auristatin F


Microbial enzyme transglutaminase




Oxyamine-auristatin F containing a short polyethylene glycol spacer




Non-native amino acids


Office of Biotechnology Products


Office of New Drug Quality Assessment










Polyethylene glycol


Polyethylene terephthalate glycol






Reverse phase high performance liquid chromatography


Single-chain variable fragment


Size exclusion column high performance liquid chromatography








THIOMAB drug conjugate


Ado-trastuzumab emtansine (Kadcyla)




Valine-citrulline cleavable linker


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All authors are employees of Pfizer, Inc and hold financial interest in Pfizer, Inc. We acknowledge Sandeep Kumar of Pfizer for the model in Fig. 1 and to our many colleagues in Biotherapeutics Pharmaceutical Science who helped review the manuscript.

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Correspondence to Roger H. Pak.

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Satish K. Singh, Donna L. Luisi and Roger H. Pak contributed equally to this work.

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Singh, S.K., Luisi, D.L. & Pak, R.H. Antibody-Drug Conjugates: Design, Formulation and Physicochemical Stability. Pharm Res 32, 3541–3571 (2015).

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