Purification of monoclonal antibodis using counter current chromatography

该项目旨在通过使用一种新的液体固定相技术，开发一种在制备、中试或工业规模上大大改进的单抗(MAbs)纯化方法。2005年，蛋白质疗法的销售额估计超过500亿美元，其中150亿美元来自抗体。据预测，抗体的增长速度将是其他蛋白质的三倍，在2010年之前的未来5年内每年增长21%，达到350亿美元以上(Datamonitor2006)。2005年有21个蛋白质大片，而2000年有6个，最后11个中有7个是抗体。这主要是因为阿斯利康、葛兰素史克和默克等大型制药公司对收购新的蛋白质药物表现出越来越大的兴趣。预计在2004年至2010年间，生物制药将占大型制药公司增长的57%。由于从2006年开始的5年内，外包生产预计将从58%增加到69%，龙沙生物公司作为最大的合同制造组织(CMO)之一，因此是生物制剂行业抗体生产过程开发和生产的主要力量，因此在抗体生产方法方面拥有既得利益。各国政府已经注意到，蛋白质疗法的供应成本很高，英国法院的几起备受瞩目的案件说明了这一点，在这些案件中，患者获得了NICE以前拒绝他们使用的药物。因此，已经有了机构牵头的项目来降低这些成本，例如欧洲的AIMS项目和FDA的21世纪倡议，这些项目试图确定并降低新生产技术的进入壁垒。这是该行业在降低内部成本方面所做的最大努力。在龙沙，已经成立了下游处理(DSP)过程工程小组，专门研究这些创新技术，而逆流提取液/液被视为该领域的关键新兴技术之一。单抗可以通过细胞发酵来生产，这是龙沙采用的方法。这些发酵液的效价一般都在5g/L以上，最高可达10g/L，单罐单抗最多可达20000L。随着这些物质到达净化过程，传统的固定床塔不再能处理施加在它们身上的负荷，需要多次循环，导致工厂吞吐量问题。这一点，再加上这种柱子的结合能力有限，意味着树脂的寿命已经减少到更少的批次。这些问题加在一起，产生了研究替代方案的重要需要。该项目旨在开发一种不同的纯化方法，一种使用液体而不是固体固定相的方法。因此，它可以在发酵过程中直接存在颗粒物的情况下运行，并且可以放大到大尺寸，而不会出现压力问题。目的是建立一种利用逆流层析(CCC)或离心分配层析(CPC)进行单抗制备规模纯化的分离方案。该协议可以使用双水相体系(ATPS)或水-有机溶剂体系。它还可以采用一种新的方法，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过程，这些方法只有在固定相为流体时才能使用。总而言之，我们的目标是使用液-液技术直接从细胞发酵中提纯单抗，每个发酵罐的规模可达200公斤。为了实现这一目标，研究项目将是广泛的，寻找一些可能的选择，尽管总是充分利用固定相的流动性质。

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.