Purification of monoclonal antibodis using counter current chromatography

该项目旨在通过使用具有液体固定相的新兴技术，开发一种在制备、中试或工业规模纯化单克隆抗体（mAb）的显著改进的方法。2005年，蛋白质治疗剂的销售额估计超过500亿美元，其中150亿美元来自抗体。据预测，抗体将显示出比其他蛋白质快三倍的增长，在到2010年的未来5年内，每年增长21%，达到超过350亿美元（Datamonitor 2006）。2005年有21个蛋白质炸弹，而2000年只有6个，最后11个中有7个是抗体。这是由于阿斯利康，葛兰素史克和默克等主要制药公司对收购新蛋白质药物的兴趣增加。预计2004年至2010年期间，生物制剂将占大型制药公司增长的57%。由于外包生产预计将在2006年起的5年内从58%增加到69%，Lonza Biologics作为最大的合同制造组织（CMO）之一，因此是生物制品行业抗体工艺开发和生产的主导力量，在抗体生产方法方面拥有既得利益。政府已经注意到，蛋白质治疗剂的供应成本很高，这已经在英国法院的几个备受瞩目的案件中得到了说明，在这些案件中，患者赢得了之前被NICE拒绝的药物。因此，有一些机构牵头的项目来降低这些成本，如欧洲的AIMS项目和FDA的21世纪倡议，这些项目试图确定和降低新生产技术的进入壁垒。这是在该行业努力降低内部成本的基础上。在龙沙，下游处理（DSP）工艺工程组已经成立，专门投入资源研究这种创新技术，并通过逆流方法使用液/液萃取被视为该领域的关键新兴技术之一。单克隆抗体可以通过细胞发酵生产，这是龙沙采用的方法。这些发酵中的滴度增加到通常超过5g/L，并且可以高达10 g/L。发酵可以高达20，000 L，其中多达200 kg mAb来自单个发酵罐。随着这些物质到达纯化过程，传统的固定床柱不再能够科普置于其上的负载，并且需要多个循环，从而导致工厂吞吐量问题。这与此类色谱柱的有限结合能力一起意味着树脂寿命已减少到更少的批次。这些问题结合在一起，产生了研究替代品的重要需求。该项目旨在开发一种不同的纯化方法，使用液体而不是固体固定相。因此，它可以在直接来自发酵的颗粒物质的存在下操作，并且可以放大到大尺寸而没有压力问题。目的是开发一种使用逆流色谱（CCC）或离心分配色谱（CPC）进行制备规模纯化单克隆抗体的分离方案。该方案可以使用含水两相系统（ATPS）或含水-有机溶剂系统。它还可以采用新的方法，例如使用离子液体，在液体固定相内使用亲和配体，或使用独特的连续逆流萃取方法，这些方法只能在固定相是流体时使用。总之，目标是使用液-液技术以高达200 kg/发酵罐的规模直接从细胞发酵中纯化mAb。为了实现这一目标，该研究项目将是广泛的，寻找一些可能的选择，虽然总是充分利用固定相的流体性质。

This project aims to develop a considerably improved method for the purification of monoclonal antibodies (mAbs) at a preparative, pilot or industrial scale by using a new, emerging technology with a liquid stationary phase. In 2005 the sales of protein therapeutics was estimated to be worth over $50 billion, of which $15 billion was contributed by antibodies. It has been projected that antibodies will show three fold faster growth than other proteins of 21% per annum over the next 5 years to 2010 reaching over $35 billion (Datamonitor 2006). There were 21 protein blockbusters in 2005 versus 6 in 2000 and 7 of the last 11 were antibodies. This is led by the increased interest shown by major pharma companies such as AstraZeneca, GSK and Merck in acquiring new protein drugs. Biologics are predicted to account for 57% of large pharma growth between 2004 and 2010. Since outsourced production is expected to increase from 58% to 69% over the 5 years from 2006, Lonza Biologics, as one of the largest contract manufacturing organisations (CMO) and therefore a predominant force in the process development and production of antibodies to the biologics sector, have a vested interest in production methods for antibodies. It has been noted by governments that the supply of protein therapeutics has a high cost and this has been illustrated by several high profile cases in the British courts where patients have won access to drugs previously denied to them by NICE. As a result there have been agency lead projects to reduce these costs, such as the AIMS project in Europe and the FDA's 21st Century Initiative, which have tried to identify and lower the barriers to entry of new production techniques. This is on top of the industry's efforts to lower such costs internally. At Lonza, the Downstream Processing (DSP) Process Engineering Group has been formed to dedicate resource to investigating such innovative technologies and the use of liquid / liquid extraction by counter current methods is seen as one of the key emerging technologies in this field. MAbs can be produced by cell fermentation and this is the approach adopted by Lonza. The titre in these fermentations is increasing to generally over 5g/L and can be as much as 10g/L. Fermentations can be as large as 20,000L with as much as 200kg mAb coming from a single fermenter. With such masses reaching the purification processes, traditional fixed bed columns can no longer cope with the load placed on them and multiple cycles are required, leading to plant throughput issues. This, together with the limited binding capacity of such columns, means that resin lifespan has been decreased to fewer batches. Combined, these issues have generated an important need to investigate alternatives. This project aims to develop a different method of purification, one that uses a liquid rather than a solid stationary phase. It can therefore operate with the presence of particulate matter direct from the fermentation, and be scaled up to large sizes without pressure problems. The aim is to develop a separation protocol for the preparative-scale purification of monoclonal antibodies using counter-current chromatography (CCC) or centrifugal partition chromatography (CPC). This protocol may use an aqueous two-phase system (ATPS) or an aqueous-organic solvent system. It may also adopt a novel approach such as the use of ionic liquids, the use of affinity ligands within the liquid stationary phase, or the use of a unique continuous counter-current extraction process, approaches that can only be employed when the stationary phase is a fluid. In summary, the goal is to purify mAbs directly from cell fermentations using liquid-liquid technology at a scale up to 200kg per fermenter. To achieve this goal, the research project will be wide ranging, looking at a number of possible options, though always taking full advantage of the fluid nature of the stationary phase.