Initial Capture

The first major purification step in a process typically involves binding the antibody to some chromatography matrix, while impurities either flow through or are differentially eluted from the antibody. Alternately, conditions can be chosen where contaminants bind, and the antibody does not.

General Affinity. The most common matrix used to bind antibodies is immobilized protein A. This Staphylococcus aureus cell surface protein binds to many, although not all, antibodies and is widely available (17,22-31). Another bacterial cell surface protein, known as protein G, has been introduced more recently. Protein G binds more types of antibodies than does protein A, and it binds them more tightly (28,29,32,33). Although more versatile than protein A due to its wider specificity, the tighter binding of protein G can cause problems, as will be described later. Both of these are widely used because of their broad specificity, wide availability, and the high degree of purification. Indeed, purities of more than 95% are readily obtained, and antibodies of this purity are usable in many applications.

Historically, there have been three problems related to the use of protein A. First, it is expensive. However, given proper care, it is possible to reuse protein A resins many times, decreasing the long-term cost (34-37). Second, early protein A resins tended to leach protein A into the product (35). This is no longer a common problem, although leaching should be tested for, especially for products intended for in vivo use (38). Protein A assays are now readily available for such testing (39). The third problem is that many antibodies require a fairly low (pH 3-4) pH to elute from protein A. There are some antibodies that cannot withstand exposure to these conditions, even if the condition is neutralized as soon as possible. Needless to say, this is an even greater problem with the tighter binding of protein G (15,17,40).

In those instances where an antibody is acid sensitive, but it is still desirable to use protein A or G, a number of potential alternative elution conditions have been reported. These include the use of basic pH conditions (41), chaotropic ions or basic pH followed by ethylene glycol (42), and peptides that mimic the protein A or G binding site (43-45).

In addition to purifying bulk antibodies, proteins A and G have other applications in the field. For instance, they can be used to remove residual whole antibody and Fc fragments in preparations of Fab and F(ab')2 fragments (17), since both proteins A and G bind to the Fc region of antibodies. Another use is subclass separation from a polyclonal antibody feedstock (e.g., serum). Although this is not always the case, often different subclasses of antibodies have different affinities for proteins A and G, which allows chromatographic fractionation of the subclasses (15,17,2223,40,46-48). Table 1 illustrates this effect. The differential binding of protein A for different subclasses is not only a blessing, but also a curse and a need for caution. One subclass that does not generally bind well to protein A under physiological conditions is murine IgG1. This is currently the most common monoclonal antibody type seen. However, this problem has been overcome, in that a number of different regimens, in particular the use of slightly alkaline binding buffers of high ionic strength, have been developed to efficiently purify murine IgG1 on protein A (49-52). The need for caution lies in that not even antibodies of the same subclass will behave exactly the same (Table 1; 40,46). Thus, although general guidelines for the binding and elution of different classes of antibodies are helpful, the protein A or G purification of each antibody must be individually optimized.

Similar natural affinity ligands exist with other specificities. Some of these are based on the fact that antibodies are glycoproteins. Examples are mannan binding protein, which targets the mannan sugars of IgM, and the lectin jacalin, which binds human IgA1 (26,49). Many others have been described in the literature (26).

Immunoaffinity. The most selective technique is that of immunoaffinity, which can take a number of forms. In the most specific, the antigen for the desired antibody, or an anti-antibody against it, is used to make a chromato-graphic resin. In a somewhat less-specific fashion, antibodies against specific types of light or heavy chains can be used. However, immunoaffinity suffers from a number of problems. First, the antigen or anti-antibody must be produced and purified. Second, binding is often so strong that very vigorous and potentially harmful elution conditions must be used (12,53-56). On a production scale, the economics of producing the antigen or anti-antibody often make this technique undesirable. Finally, for those antibodies intended for in vivo use, assay techniques must be available to show that the product is free of antigen or anti-antibody. Furthermore, unless process steps downstream of the immunoaffinity column provide adequate viral clearance, as shown by validation studies, the ligand used to make the immunoaffinity support will need to have been purified from a process validated to remove viruses (1). Also, it must be demonstrated that either the product is free of any leached ligand (as noted above) or the ligand does not affect product safety (1). These additional requirements would add significantly to the cost of immunoaffin-ity chromatography.

A renaissance of this technique may come from the advent of chemically and genetically engineered pseudoan-tigens. Developed for specificity and reasonable binding affinity and produced by recombinant or chemical methods, these compounds (often small peptides) could overcome most, if not all, of the earlier objections (57). A similar case is the development of mimics of protein A and of generic anti-antibodies (58,59). Generic anti-antibodies are those targeted against constant regions of the antibody, making

Table 1.

Percentage of Given Antibody Eluted at Given pH

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