Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies

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Abstract

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1 mg/ml human immunoglobulin G (IgG), 10 mg/ml serum albumin and 2 mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101 ± 7%, a purity of 99 ± 0% and a yield of native IgG of 97 ± 4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.

Introduction

Antibodies constitute the first line of defence of the body immune system. They are naturally produced by a type of white blood cells known as B-cells, which are able to specifically recognise and bind foreign and potentially toxic molecules or pathogens such as bacteria or viruses. When monoclonal antibodies (mAbs) were first described by Köhler and Milstein, in 1975 [1], it was immediately recognised that they had great potential as powerful and specific therapeutic agents. Presently, the US Food and Drug Administration (FDA) has approved 21 monoclonal antibodies to treat different types of cancer (e.g. breast cancer, non-Hodgkin's lymphoma, leukaemia, colorectal cancer, prostate cancer), transplant rejection, auto-immune diseases (rheumatoid arthritis and Crohn's disease), asthma, cardiovascular and infectious diseases [2].

The production of therapeutic antibodies must meet high standards of safety, efficacy and potency, which translates into very high purity levels [3]. Impurities including host cell proteins, DNA, culture media constituents and potential contaminants such as endotoxin and viral particles must be removed [4]. In addition, because many of the diseases treated by mAbs require high doses or chronic administration, economical process-scale production of these molecules is critical. Downstream processing has been considered the bottleneck in providing mAbs at reliable costs. The basic steps for the downstream processing are (i) clarification by removal of cells and cell debris by centrifugation or microfiltration, (ii) concentration by ultrafiltration, (iii) selective purification steps, (iv) virus inactivation and removal and (v) validation and quality control tests [5]. Clarification, concentration and virus clearance contributes only with about 10% of the total downstream costs. Therefore, the major potential for optimisation is found in the selective purification steps.

Nowadays, a new production process is developed for each MAb candidate. This approach will however no longer be suitable when the mAbs candidates currently in pre-clinical and clinical trials reach the process development stage. A generic and efficient recovery and purification technology on a large scale is therefore of great importance. The downstream processing costs of mAbs are often dependent on the culture medium components, such as albumin, transferrin, insulin and other serum proteins.

Partitioning in aqueous two-phase systems (ATPSs) has proved to be a valuable tool for separating and purifying mixtures of biomolecules by extraction. Moreover, ATPSs are an ideal technology where clarification, concentration, and partial purification can be integrated in one step. This technique can be made highly selective and can be suited for continuous operation in large scale, thus allowing wider biotechnological applications.

Both polymer/polymer and polymer/salt ATPSs have advantages over other commonly used separation and purification techniques. Since the bulk of both phases consists mainly of water (80–90%) and most polymers have a stabilising effect on the protein tertiary structure [6], ATPSs form a gentle environment for biomaterials such as proteins. In addition, the interfacial tension is extremely low, creating a high interfacial contact area of the dispersed phase and thus an efficient mass transfer [7]. Furthermore, this process is cost-effective and its scale-up is very easy and reliable [8].

Aqueous two-phase systems are hence a promising technique and have been successfully used to isolate antibodies from hybridoma cell supernatants [9], [10]. It is also a powerful tool for antibody analysis, namely for purification and fractionation, detection and separation of conformational isomeric forms, examination of surface properties related to antigen specificities and for providing new interesting information about the events occurring upon antigen–antibody complexation and about possible ligand-induced conformational changes [11]. Nevertheless, the application of ATPSs to process-scale has been hindered because the mechanisms of partition are still poorly understood and method development is wholly empirical [12].

The aim of this work is to investigate the feasibility of using ATPSs to recover human IgG from a protein mixture, containing albumin and myoglobin, to optimise this separation and to identify the key variables that rule this partitioning.

Section snippets

Chemicals

Polyethylene glycols (PEG) with molecular weight of 1000, 6000, 10,000 and 20,000 were obtained from Fluka (Buchs, Switzerland), and PEG 3350 from Sigma (St. Louis, MO, USA). All polymers were used without further purification. Potassium phosphate dibasic anhydrous (K2HPO4), sodium phosphate monobasic anhydrous (NaH2PO4) and sodium chloride (NaCl) were purchased from Sigma. Human IgG for therapeutic administration (product name: Gammanorm) was purchased from Octapharma (Lachen, Switzerland), as

Proteins characterisation

One of the factors that affects the partition of proteins between two phases is their physical and chemical properties, namely the size, charge and hydrophobicity of the molecule. Furthermore, since it is the exposed groups of the surface that come in contact with the phase components, partition can be regarded as a surface-dependent phenomenon [8]. The larger the size of the protein, the larger the exposed surface that can interact with the surrounding phase components.

The three-dimensional

Conclusions

The feasibility of using ATPSs for the purification of human IgG from a mixture of proteins containing human serum albumin and myoglobin is shown in this paper. A central composite design allowed the definition of appropriate models for the purification parameters KP, YTop and YNat_IgG, which in turn led to the definition of the best purification conditions. Accordingly, it is possible the selectively recover IgG in the top phase with high yield and purity using high concentration of NaCl and

Acknowledgments

This work was carried out within the European project AIMs (contract no. NMP3-CT-2004-500160), supported by funding under the Sixth Research Framework Programme of the European Union. P.A.J.R. and A.M.A. acknowledge Fundação para a Ciência e Tecnologia for a PhD fellowship (BD 25040/2005) and post-doctoral fellowship (BPD 18931/2004), respectively.

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