Integrated Downstream Processing

Over the last decades, the pharmaceutical industry has invested a tremendous amount of resources and capital into the development of continuous processes to improve process efficiency and robustness. Increasing expression levels in modern cell culture and implementing continuous upstream processes has caused the downstream purification to become the “bottleneck” in the manufacturing of biopharmaceuticals, especially of monoclonal antibodies. New downstream approaches are required in time and cost effective ways to purify biomolecule’s while retaining the protein’s characteristics. Approaches such as integrated downstream processes with multi-column continuous chromatography show promising results with increased throughput and reduced capital and operational expenses. Recently, the first integrated continuous downstream process at the production scale was reported by Amgen [1]. This White Paper highlights the major advantages and remaining barriers when implementing these process approaches into the downstream purification scheme. In the first part of this paper, the focus is on the process development aspects. Guidelines for the development of robust integrated continuous processes are discussed. Examples for multi-column continuous chromatography and inline buffer adjustment approaches are introduced. Next, guidelines are presented on how overcome technical barriers in the regulated environment of the pharmaceutical industry. The appropriate equipment designs, applicable automation and analytical tools suitable for multi-column chromatography, as well as their proper risk assessments are described. Although, when implementing these approaches the initial research and capital investment costs are significant, the manufacturing costs decrease and risks are mitigated due to smaller equipment. Ultimately, this leads to a more robust, yet flexible processes. By minimizing complexity of the integrated continuous downstream processes, additional benefits are obtained. © LEWA Process Technologies, 2015 02 Integrated Downstream Processing Downstream Processing Since introducing for the manufacturing of recombined insulin in the 1980’s, a tremendous amount of resources have been investigated in the development, scale-up and optimization of bio-pharmaceutical processes, in particular, in the downstream purification processes (DSP). These processes are an essential part of the manufacturing of polypeptides, proteins, enzymes, and monoclonal antibodies (mAb). A typical DSP scheme, as shown in Figure 1 for mAb purification, includes capture, purification and polishing steps. Different chromatographic modes are applied such as affinity, ion exchange, size exclusion and hydrophobic interaction chromatography to purify complex cell culture mixtures containing very closely related but also very different molecules. The chromatographic steps process usually the complete batch in one or two injections onto large LPLC columns requiring large tank systems. Between chromatographic steps, there are filters and membranes for viral clearance or buffer exchange, as well as large hold-up tanks that are also used for viral inactivation. Membranes and filters are operated continuously by installing parallel units. Figure 1 Scheme of generic batch DSP for mAb [2] Approximately 15 years ago, research started on implementing continuous chromatography into the DSP to improve the process efficiency and reduce equipment sizes [3-19]. At that time, multi-column continuous (MCC) chromatography such as the simulated moving bed (SMB) technology became an established separation tool for synthetic molecules [20-24]. Some of the early research in the field of continuous DSP were conducted at the ETH Zurich, University of Magdeburg and Purdue University [10-13, 15, 17]. Due to technical and assumed regulatory barriers, MCC processes were not adapted into the bio-manufacturing and only picked up again 5 years ago [25 43]. Initially, upstream processes were the “bottleneck” when titers were less than 1 g/L of mAb. Over the last decade, the upstream expression levels rose above 10 g/L. Downstream became © LEWA Process Technologies, 2015 03 Integrated Downstream Processing the “bottleneck”, especially first capture step. Resins such as Protein A are expensive and have limited lifetime. Another important driver is the introduction of bio-similar and bio-better to the market, in particular, with the first approval of Remsima in 2012 by Celltrion [44]. New downstream approaches are now required that are time and cost effective and retain physical, chemical and biological integrity of molecules. Industrial and academic research teams are re-examining approaches such MCC chromatography integrated into the continuous DSP as shown in Figure 2. Figure 2 Scheme of generic integrated continuous DSP for mAb [2] This integrated platform is now a closed system with limited or no hold points. Not only the missing tanks reduce the footprint but also the smaller equipment scale due to the continuous operation. The smaller sizes enable also the implementation of single-use technology [37, 38, 42, 45, 46]. All these efforts together simplify the cleaning procedures and process validations; and, therefore, shorten the turn-around time between batches. This is especially important for CMO’s. Replacing batch chromatography with MCC chromatography as shown in Figure 3 [18] improves the process efficiency and robustness , reduces the buffer consumption and decreases the equipment size used for the chromatographic step. , Increased throughput and packing utilization have been demonstrated at the bench-top scale, which reduce equipment and manufacturing costs; however, the full potential of the integrated continuous DSP platform is reached when both the capture and IEX chromatographic steps (see Figure 2) are operated continuously. [30-36]. As shown on the left of Figure 3, columns operate in a parallel flow direction. During one switch, each column fulfills a particular task of the separation recipe. This is in contrast to the sequential execution of the recipe on a large column as explained in Figure 4. After switching the column in the flow direction, tasks are changed based on the recipe. Due to the switching, © LEWA Process Technologies, 2015 04 Integrated Downstream Processing the process can be continuously fed, one column at a time. This process has no internal buffer recycling. Significant buffer reduction can be obtained in a true counter-current processes. Such a process is shown on the right of Figure 3. A regeneration section for cleaning and reequilibration is added to the conventional SMB process. Figure 3 MCC chromatography: left – parallel operation, and right – partial counter-current operation [18] To guarantee the success of the integrated operation, each DSP step must be scaled and scheduled accordingly to avoid any hold-points or idle times. Some single process units can easily operate continuously such as filtration and buffer-inline adjustment steps (see Section: Inline-Buffer-Adjustment). Traditionally, chromatographic processes are run in the batch mode. If they are transferred into the continuous mode, products eluting of the column have no uniform characteristics but a cyclic one which changes composition with time. These behaviors can be observed in the cyclic characteristic of capture processes from GE HealthCare [31], NovaSep, Inc. [25] and ChromaCon [43]. Multi-column continuous chromatography applications and the technical challenges [2, 4042] are in the design of the chromatographic skid, the valves and the chromatographic columns, as well as in the implementation of process analytical tools (PAT). These challenges are weighted with the high initial capital investment for the skid, multiple pumps and columns, as well as more of a complex process design working in a regulated environment. For each of the chromatographic steps, comprehensive risk assessment and control as well as for the entire DSP are required. Some of the inherent risks of the traditional batch DSP are reduced due to the continuous operation. For instance, the entire batch is not lost in the event of a failure, as happens during single one-time injections into large column. Other risks are enhanced but can © LEWA Process Technologies, 2015 05 Integrated Downstream Processing be mitigated by implementing control strategies [30, 47, 48] using PAT such as a guarantee of the stability of the bio-molecules during long-term operation. When connecting process units to an integrated continuous DSP scheme, process automation systems control not only the single processes but also the complete DSP scheme. Based on the critical process parameters (CPPs) and critical quality attributes (CQAs) of the processes, sensors and detectors are selected to monitor the process parameters and quality attributes in the required ranges during continuous operation. The process parameters for each downstream process (DSP) step are defined during the process development phase. Parameters such as flow rates depend on the process throughput requirements, upstream operations and batch sizes. Based on these parameters and pressure limitations of the resins and membranes, the size of the pumps, valves, pipes, and columns can be determined. In case of an integrated continuous DSP process (see Figure 1), the scale of sequential process units depends on their previous process steps. The major chromatographic processes are connected using inline-buffer adjustment systems that are described in the section below. Surge tanks are installed as a risk mitigation measures to avoid issues arising from the cyclic collections of the previous product streams and/or unexpected delays. Until the recent announcement of Amgen [1] there has not been a reported case of continuous DSP at the production scale. Barriers remain by implementing these innovative approaches into the downstream processing. With its high degree of complexity,