Fragment antigen binding domains, or F(ab) fragments, are derived from the upper branches of the ‘Y’-shaped antibody molecule. They form the antigen binding interfaces of intact antibodies, known as the paratope, and retain their inherent target binding capacities even when isolated from the rest of the protein (the Fragment crystallizable or Fc fragment). Separation can be achieved enzymatically using an either papain or pepsin. Cleavage with papain yields two individual F(ab) fragments consisting of a single light chain and half of a heavy chain (comprising a VH and CH1 region), whereas cleavage with pepsin produces F(ab')2 – two individual F(ab) fragments joined together by a disulfide bond. Additionally, modern molecular biology techniques have enabled the genetic engineering and production of F(ab) fragments in host cell systems, allowing them to be expressed independently of the Fc region without the need for enzymatic cleavage and permitting an array of different conformations.
Advantages
The ‘downsizing’ of the antibody molecule to either F(ab) and F(ab′)2 fragments offer several potential advantages over their full-size counterparts due to alterations in their physiochemical properties. For instance, due to their lack of glycosylation and relatively small size, antibody fragments may prove easier and less costly to manufacture as they can be produced in prokaryotic expression systems such as E. coli. Furthermore, the reduced size of fragments permits deeper tissue penetration compared to full-size antibodies, in some circumstances facilitating entry to tissues completely impenetrable to the latter [1]. This makes them attractive to drug developers, especially those looking to gain further access to solid tumors.
Half-life flexibility
F(ab) and F(ab′)2 fragment antibodies also offer a way of eliminating non-specific binding between antibody Fc regions and Fc receptors on cells (which are commonly found on immune cells such as macrophages, dendritic cells, neutrophils, NK cells and B cells). The lack of an Fc domain in these molecules also has additional impact on their metabolism in the body. F(ab) and smaller fragment antibodies have the shortest circulating half-lives, whereas F(ab′)2 fragments stay active in the body for longer; however, F(ab′)2 fragments still have much shorter half-lives than full-size antibodies. These varying half-life lengths provide researchers with the means to tailor the duration of a drug response. For example, a F(ab′)2-based antivenom proved to be a more optimal treatment for pit viper envenomation than a F(ab)-derived version, which represented the current treatment standard at the time of the clinical study [2]. The F(ab′)2 form of the antivenom treatment was more stable and stayed in the patients’ bloodstreams long enough to reduce the risk of bleeding due to coagulopathy caused by snake venom, even without the need for maintenance doses [2]. The authors of this study also noted that immune reactions were markedly less problematic with F(ab) and F(ab′)2 than those with the full-length antibodies, suspecting this was due to the lack of the Fc portion, which is purported to cause the most adverse reactions [2].
Better imaging
Medical and research imaging are other areas where the use of F(ab′)2 fragments can offer clinicians and researchers potential benefits. Because of their pharmacodynamics, F(ab′)2 antibodies show a faster clearance of background signal from host subjects than full-length antibodies do, but achieve greater absolute uptake at the target site than smaller fragments. This reduces the wait time between antibody tracer administration and imaging (which can be several days or more in some cases) and increases the accuracy of imaging results.
Purification hurdles
As F(ab′)2-based therapeutic and diagnostic molecules progress along the drug development pathway, it makes more sense to express them in host cell systems at larger scales of production, as enzymatic cleavage adds an extra processing step that becomes more inefficient as volumes increase. However, many host cell systems, such as Chinese Hamster Ovary (CHO) cells, when engineered to express recombinant F(ab′)2, also express F(ab) fragments, even despite attempts to eliminate F(ab) expression through additional protein and cell line engineering. Therefore, purification strategies need to be on the money to ensure that a quality product with a low contamination profile is obtained. This represents a challenge for two main reasons: first, F(ab′)2 fragments (in addition to smaller F(ab) fragments), unlike their full size counterparts, cannot always be purified effectively using protein A- or protein G-based chromatography resins due to their lack of the Fc region (which possesses the main moiety that interacts with these media). Second, F(ab′)2 and F(ab) fragments are almost identical, presenting the purification scientist with another conundrum.
Purification solutions
Protein G retains a low affinity for the CH1 domains of F(ab′)2 and F(ab) fragments, so it is worth trying protein G-based resins at the exploratory and upstream processing stages of F(ab′)2 antibody development, before trying more expensive solutions. If F(ab′)2 recovery rates are below acceptable standards with protein G affinity purification, there are alternative resins researchers can try. A lot of these options are actually modified versions of protein G, designed to interact specifically with F(ab) domains. In this regard, look out for any resins that bind to either the VL or CL domains. As this first step uses affinity chromatography, individual F(ab) fragments will co-purify with F(ab′)2. Therefore, after isolating the F(ab′)2/F(ab) fraction from the rest of the crude sample, it is essential to put further purification steps in place that will separate these two similar antibody species. A two column-process comprising a cation exchange resin followed by a ceramic hydroxyapatite polishing resin can work well here. F(ab) should dissociate from the cation exchange column during the elution step before the F(ab′)2 portion, and the ceramic hydroxyapatite should separate out any remaining impurities due to its multi-modal properties. In fact, using this approach you may not even require an affinity chromatography step at all in the first place. Reducing the number of steps in your process in this way will serve to ensure your workflow is readily scalable, while maintaining product purity and cGMP compatibility.
Concluding remarks
F(ab')2 fragment antibodies can be used for a variety of different applications, both in the research lab and in the clinic. They offer another gradation in the duration of clinical responses and can reduce time windows in medical and research imaging, in addition to several other benefits such as lower production costs and potentially reduced risks of adverse reactions. Producing high-quality batches of these antibodies that are substantially free of F(ab) can be challenging, but with the right workflow in place, developed in a step-wise and considered manner, this task is not impossible.
References
[1] Nelson AL. Antibody fragments: hope and hype. MAbs. 2010 Feb;2(1):77–83. [PMID: 20093855]
[2] Bush SP, Ruha A-M, Seifert SA, Morgan DL, Lewis BJ, Arnold TC, et al. Comparison of F(ab’)2 versus Fab antivenom for pit viper envenomation: a prospective, blinded, multicenter, randomized clinical trial. Clin Toxicol (Phila). 2015 Jan;53(1):37–45. [PMID: 25361165]
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