INTRODUCTION

One of the major dilemmas associated with the production of r-DNA proteins for human consumption is the extraction and purification from very crude feed stocks.

In principle, such recombinant proteins should be purified to homogeneity, so as to avoid undesired side effects when they are injected with traces of protein impurities that can be immunogenic, especially if derived from host cell proteins (HCPs) of non-human origin. In practice, even under the best conditions, it is very laborious to achieve purity levels better than 99% [1, 2]. The last purification step, for removing remaining impurity traces, is generally prohibitively expensive and leads to considerable loss of bio-pharmaceutical product. This is the reason why a protein purification process design for biopharmaceuticals starting from crude feed stocks is relatively complicated; it involves several unitary separation steps and implies to consider important parameters such as the relative amount of the target protein in the crude extract, its physicochemical properties and the desired final purity goal. The analysis of these properties is at the basis of the selection of chromatographic resins having orthogonal properties so as to increase the separation efficiency from one step to another. The first chromatographic separation step of the process is based on the selectivity of the resin for the target protein. By that way it is possible to concentrate the protein of interest and to get rid of the maximum amount of protein impurities. This step is generally named

“capture” [3]. If the capturing resin is based on a biological affinity ligand, the resulting purification factor in a single step could be very substantial. This is the case of antibody capture using a Protein A column [4] where the purity of the target protein can go up to 95-98%.

The pre-purified protein from the capture phase is then submitted to an intermediate separation process capable of producing fractions where the protein of interest is collected in one or two of them [5-8]. Resins used for this purpose are regular or high capacity ion exchange chromatography (IEC) as well as hydrophobic interaction chromatography resins (HIC). In spite of a stringent selection of chromatographic conditions for the first two steps, the final purity of the target proteins from the second step is never perfect; it contains traces of a number of protein impurities coming from the expression system or from the

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biological crude extract. In order to reach purity levels capable to meet regulatory requirements it is necessary to add at least another step called “polishing”, able to eliminate all of the remaining impurity traces. For accomplishing this task the ideal chromatographic resin should be capable of adsorbing all impurities and leaving the target protein in the flowthrough. This configuration would allow reducing the size of the column as compared to a situation in which the target proteins would be adsorbed specifically and impurities eliminated in the flowthrough. Such a definition corresponds to an affinity column; unfortunately protein traces to be removed are constituted of a large number of species and bioaffinity adsorbents corresponding to classical definitions are unpractical. Considering the above context, the most popular approach for polishing is designed around gel permeation supports based on molecular size discrimination [9-11]. Most of the times gel permeation appears as the best choice, because it is orthogonal towards the previous separation steps. However, it suffers from a number of productivity issues that limit the applicability at preparative large scale. In a large number of cases gel filtration had proven good performance in separating impurities essentially because they were of different molecular size. Nevertheless mass similarities between the target protein and impurities create a situation that cannot be resolved by this approach.

Other methods have also been described, based either on HIC [12, 13] or IEC [14, 15]; both methods are process-specific and must be utilized under the best possible conditions for accommodating the adsorption of most of the impurities and the collection of the target protein in the flowthrough.

In the attempt to find a generic process for the removal of impurity traces, a new approach based on the use of a multitude of combinatorial hexapeptide ligands immobilized on polymethacrylate beads (one ligand structure per bead) is reported in this chapter. It demonstrates the ability of the Equalizer Beads to capture impurities yielding the target proteins significantly purer than before the treatment. This approach has been first applied to the polishing of pure sperm whale myoglobin artificially contaminated with either albumin-depleted human serum proteins or E. coli protein whole extract, in order to validate the polishing method, after that it has been used for removing low abundance impurities from three tentatively pure protein preparations: recombinant Protein A, expressed in 116

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Escherichia coli and supplied as 99% pure by the manufacturer, human serum albumin (HSA), expressed in Pichia pastoris and supplied as a >96% pure product, and injectable monoclonal antibodies (Mabs), produced by hybridoma cells cultured in either standard or protein-free culture medium.

4.1.1. THE “EQUALIZER BEADS”: THEORY AND FUNCTIONING

Affinity adsorption is the most selective way for capturing single proteins from very crude dilute feed stocks, adding concentration to a purification effect. After the binding capacity of the solid phase is reached, the process is stopped, the adsorbed protein is recovered and a second cycle can be activated. If this simple mechanism is extended to a large number of different affinity solid phase ligands mixed together, many proteins would be captured at once by their own complementary ligand. Each unique affinity ligand within the pool would bind and concentrate its specific protein partner up to the point of ligand saturation. If in such a mixed bed one loads a complex mixture composed of many proteins, one of them being largely dominant (e.g. 99% purity), this latter will saturate its corresponding solid phase ligand very rapidly, while impurity traces will not. Using loading conditions where the impurities do not saturate the solid phase ligands, the flowthrough of the column will be composed of the dominant protein of purity substantially higher than before the process. Therefore, this process can be used for the removal of impurity traces at the end of downstream separation processes.

It is obvious that the bottleneck of this approach consists in the possibility of producing a sufficiently ample spectrum of different affinity ligands. And in fact no one had ever been able to achieve this purpose, until a few years ago, when Thulasiraman and co-workers [16] described the synthesis of solid-phase ligand libraries of huge diversity via the “split, couple, recombine” combinatorial chemistry [17-19]. In order to produce a sufficient diversity of affinity ligands, a library of 64 millions of hexapeptides (made by combining the 20 natural amino acids in all the possible ways) was synthesised on polymethacrylate beads by Peptides International (Louisville, Kentucky). Each bead, with an average diameter of 65 μm, carries about 50 pmoles of the same hexapeptide distributed throughout its core. The functioning mechanism of such a technology operated 117

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either in overloading or not overloading conditions (the latter being the one required for achieving a highly purified protein in the flowthrough) is depicted in figure 4.1 (panels A and B, respectively, for the two conditions).

A

Figure 4.1. Principle of functioning of the Equalizer Bead technology in overloading (A) or not overloading (B) conditions. Samples are first incubated with the Equalizer Bead library (a), and specific recognition takes place (b). Next the unbound proteins are washed away (c) and the equalized sample are finally recovered from the beads.

a) Incubation

b) Specific recognition

c) Washes

Equalized proteins High

abundance proteins

a) Incubation

b) Specific recognition

c) Washes

Impurities Highly

purified protein

B

d) Elution d) Elution a) Incubation

b) Specific recognition

c) Washes

A

Equalized proteins High

abundance proteins

a) Incubation

b) Specific recognition

c) Washes

Impurities Highly

purified protein

d) Elution d) Elution

B

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