Bioconjugation chemistry is the joining of biomolecules to other biomolecules, small molecules, and polymers by chemical or biological means. This includes the conjugation of antibodies and their fragments, nucleic acids and their analogs, and liposomal components (or other biologically active molecules) with each other or with any molecular group that adds useful properties. These molecule groups include radionuclides, drugs, toxins, enzymes, metal chelates, fluorophores, haptens, and others.^{1-3, 7-9}

The conjugation of monoclonal antibodies for therapeutic purposes is currently undergoing a flurry of research and development activity.^2,11-12 These developments may not only change the way drugs are delivered, but could lead to safer protocols by dramatically reducing the dose to achieve efficacy without causing harmful side effects. Monoclonal antibodies can be utilized for drug or isotope deliveries, but as the biological functions of monoclonal antibodies are often sensitive to subtle variations in structure, attention must be paid to the chemical aspects of preparing, purifying, and characterizing these conjugates. The most commonly employed method for covalently crosslinking monoclonal antibodies to other molecules is the use of special reagents.

This article will primarily focus on the experimental design, protocols, and procedures for the preparation of in vivo therapeutic drug conjugates, such as the conjugation of monoclonal antibodies to other molecules using ligand and metal chelates, toxins, cancer drugs, and other proteins under cGMP or general large-scale manufacturing protocols. It focuses only on the chemical modification of the antibodies. Preparation of antibody fragments such as Fab' and F(ab')₂ via enzymatic processes will not be discussed. For more information on the preparation of antibody fragmentation, see Hermanson (pages 478-482).¹

This article will address the following specific topics: reactive groups of proteins (specifically, monoclonal antibodies) that are available for modification, including their naturally occurring amino acids and reactive groups introduced by chemical modification; reagents that can be used to couple molecules; the reaction environment; the current status of experimental procedures used in laboratory preparation; purification and isolation of the conjugates; storage; and manufacturing.

Figure 1. A generic scheme for bioconjugation reaction

Amines (lysines, α-amino groups). One of the most common reactive groups of proteins is the aliphatic ε-amine of the amino acid lysine. In general, nearly all antibodies contain abundant lysine. Lysine amines are reasonably good nucleophiles above pH 8.0 (pK_a = 9.18)⁴ and therefore react easily and cleanly with a variety of reagents to form stable bonds (Equation 1).

Antibody-NH₂ + Z-B →

Antibody-NHB + Z-H (1)

Other reactive amines that are found in proteins are the α-amino groups of the N-terminal amino acids. The α-amino groups are less basic than lysines and are reactive at pH~7.0. Some of them can be selectively modified in the presence of lysines. Since either N-terminal amines or lysines are virtually always present in antibodies, and since they are easily reacted, these aliphatic amines provide the most commonly employed method of antibody modification.

Thiols (cystines, cysteine, methionine). Another common reactive group in antibodies is thiol residue from the sulfur-containing amino acid cystine and its reduction product cysteine (or half cystine). Cysteine contains a free thiol group, which is more nucleophilic than amines and is generally the most reactive functional group in a protein. Thiols, unlike most amines, are generally reactive at neutral pH, and therefore can be coupled to other molecules selectively in the presence of amines (Equation 2). This selectivity makes the thiol group the linker of choice for coupling proteins. Methods that only couple amines (for example, amine reaction with gluteraldehyde) can result in formation of homodimers, oligomers, and other unwanted products (see Hermanson, pages 470-472).¹

NH₂-Antibody-SH + Z-B →

NH₂-Antibody-SZ + BH (2)

Since free sulfhydryl groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide groups. Immunoglobulin M is an example of a disulfide-linked pentamer, while the subunits of Immunoglobulin G are bonded by internal disulfide bridges. In such proteins, reduction of the disulfide bonds with a reagent such as dithiothreitol (DTT) is required to generate the reactive free thiol. However, this method also splits the chain linkage in these antibodies and reassembly of the chains to allow proper folding may not be possible. In addition to cystine and cysteine, some proteins also have the amino acid methionine containing sulfur in a thioether linkage. Selective modification of methionine is generally difficult to achieve and is seldom used as a method of attaching drugs and other molecules to antibodies. The literature describes the use of several thiolating crosslinking reagents such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA), and sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (Sulfo-LC-SPDP) to provide efficient ways of introducing multiple sulfhydryl groups via reactive amino groups. ^{1-3, 8}