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5.1 INTRODUCTION

Chapters 3 and 4 investigated how to analyse Fv expression levels in E. coli cultures during the early stages of process optimisation and during scale-up. However, it is also critically important that Fv, thus produced, can be recovered from the culture in an active and usable form. The present chapter addresses this issue.

Conventional monoclonal antibodies may be conveniently recovered from cultured media using affinity adsorbents comprising protein A (Coding, 1980) or protein 0 (Akerstrom et al, 1985). Since neither of these reagents bind to Fv fragments (Derrick and Wigley, 1992) a different approach is required. A popular approach is to tag the Fv of interest with a polyhistidine tail and then to recover it from cultured media by immobilised metal affinity chromatography (IMAC). There has been a lot of research in this field (Skerra et al, 1991; Essen and Skerra, 1993). The technique has the advantage of being generic since it is relatively straightforward to graft a polyhistidine tail onto any Fv; however, there are also some disadvantages. These include: the potential for proteins other than Fv, but which have histidines exposed at their surface, to bind the column and therefore to contaminate the recovered Fv; and the technique's inability to discriminate readily between active and inactive Fv. [For many applications, it will be preferable only to have active Fv in a final product. For example, in the application opportunities discussed in Chapter 1 - such as separation technology - the presence of inactive Fv would at best result in reduced capacity and may result in deleterious effects such as non-specific binding.]

Another popular approach for recovering Fv from culture is antigen affinity chromatography (Riechmann et al, 1988; Anthony et al, 1992; King et al, 1993). This technique has the attraction of being very specific for active Fv in the binding-phase; however the operator is then left with the problem of how to release the Fv from the adsorbent. If too strong an eluant is used the Fv may be denatured, resulting in some inactive Fv in the recovered fraction. If too weak an eluant is used, the Fv will be

inefficiently released or not released at all. In the previous chapter, an eluant of pH 2.5 buffer was selected by trial and error; this resulted in the recovery of an Fv preparation with very good immunoreactivity (87%). Similarly, a leading group at Celltech elected to desorb one of their Fvs from an antigen affinity column with lOOmM citric acid (which also has a pH of approximately 2.5); refer to King et al, 1993. However, it seems unlikely that it will be possible to find suitable buffers by trial and error for all Fvs, particularly high affinity Fvs. Furthermore, the problem will become much worse upon scale-up when the Fv will be exposed to the desorption buffer for longer due to the larger void-volume of process-scale columns. Clearly, a more rational design of eluants is required.

Some progress towards the rational design of eluants for antigen affinity chromatography was made by Anthony et al (1992) who purified an Fv specific for digoxin on an immobilised antigen analogue, ouabain. Anthony recovered bound Fv from the adsorbent with a 20mM solution of the same analogue. In this chapter, the potential of using immobilised antigen analogues was investigated in more detail. The example used was an Fv ("Fv 4155") which has a primary specificity for the hapten estrone-3-glucuronide (a urinary metabolite of estriol, a steroid hormone with a role in human fertility). This Fv was used because the monoclonal antibody from which it is derived is known to bind to two analogues of its hapten with progressively lower affinity (estriol-3-glucuronide and estrone). This offers the intriguing prospect of making an affinity column comprising immobilised estrone and eluting with a solution of estriol-3-glucuronide made up in a buffer of neutral pH. It was hypothesised that such an eluant might release Fv from the column very efficiently as estriol-3- glucuronide has a higher affinity for the Fv than the analogue on the column. Moreover, the eluant would not be expected to denature the Fv as can be the case with eluants comprising buffers of extreme pH. However, the proposed process would leave the purified Fv complexed with the second steroid, estriol-3-glucuronide. Therefore, an important objective of this study was to determine whether this Fv- complex could still function as an active species; for example, would it still be able to bind to native antigen in an immunoassay?

This chapter describes the synthesis of an affinity column comprising estrone and its use to recover Fv 4155 from E.coli culture supernatant. The amount of active Fv 4155 that could be recovered from the column by eluting with estriol-3-glucuronide was compared with the amount that could be recovered with "conventional" elution conditions (i.e. acidic buffer). Since this particular Fv can be produced with a myc tag (grafted onto the C-terminus of its chain) without any apparent deleterious effects on expression levels or activity, it was possible to monitor active Fv 4155 throughout the recovery process by using an ELISA comprising the myc-specific tracer antibody described by Ward et al. (1989). However, the "motif assay" (as described in chapters 3 and 4) could not be used to monitor total Fv throughout the process because the motif-specific antibodies do not recognise Fv 4155 in complex with estriol-3-glucuronide. Instead total protein was monitored using a colorimetric assay (Bradford, 1976). A further objective was to investigate the storage stability of the Fv recovered in complex with estriol-3-glucuronide. It was hypothesised that the Fv complex may have improved storage stability compared with native Fv as it is "locked-in" to an active configuration.

5.2 METHODS

5.2.1 Selection of antigen analogues

The fine specificity of the 4155 parent monoclonal antibody and its cross-reactivity profile with related steroids was worked out by a collaborating Colworth scientist. Dr. Coley, using the method published in Gani et al (1994). This was used to select a ligand and an eluant. In brief, the lowest affinity analogue (estrone) was chosen as ligand, and a mid-range affinity analogue (estriol-3-glucuronide) was chosen as an eluant. See Fig 5.1.

5.2.2 Svnthesis of estrone-agarose affinitv column

Estrone was coupled via its hydroxyl to a chromatography medium (Epoxy 6B

Sepharose, Pharmacia). This product has a convenient 12-atom spacer arm. The coupling protocol was as detailed below :-

Fig 5.1: Steroid analogues used to purify