Scholarly article on topic 'Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements'

Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Xhorxhi Gjoka, Rene Gantier, Mark Schofield

Abstract The goal of this study was to adapt a batch mAb purification chromatography platform for continuous operation. The experiments and rationale used to convert from batch to continuous operation are described. Experimental data was used to design chromatography methods for continuous operation that would exceed the threshold for critical quality attributes and minimize the consumables required as compared to batch mode of operation. Four unit operations comprising of Protein A capture, viral inactivation, flow-through anion exchange (AEX), and mixed-mode cation exchange chromatography (MMCEX) were integrated across two Cadence BioSMB PD multi-column chromatography systems in order to process a 25L volume of harvested cell culture fluid (HCCF) in less than 12h. Transfer from batch to continuous resulted in an increase in productivity of the Protein A step from 13 to 50g/L/h and of the MMCEX step from 10 to 60g/L/h with no impact on the purification process performance in term of contaminant removal (4.5 log reduction of host cell proteins, 50% reduction in soluble product aggregates) and overall chromatography process yield of recovery (75%). The increase in productivity, combined with continuous operation, reduced the resin volume required for Protein A and MMCEX chromatography by more than 95% compared to batch. The volume of AEX membrane required for flow through operation was reduced by 74%. Moreover, the continuous process required 44% less buffer than an equivalent batch process. This significant reduction in consumables enables cost-effective, disposable, single-use manufacturing.

Academic research paper on topic "Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements"

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Journal of Biotechnology

journal homepage: www.elsevier.com/locate/jbiotec

Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements

Xhorxhi Gjoka, Rene Gantier, Mark Schofield *

Pall Life Sciences, 20 Walkup Dr., Westborough, MA, USA

ARTICLE INFO ABSTRACT

The goal of this study was to adapt a batch mAb purification chromatography platform for continuous operation. The experiments and rationale used to convert from batch to continuous operation are described. Experimental data was used to design chromatography methods for continuous operation that would exceed the threshold for critical quality attributes and minimize the consumables required as compared to batch mode of operation. Four unit operations comprising of Protein A capture, viral inactivation, flow-through anion exchange (AEX), and mixed-mode cation exchange chromatography (MMCEX) were integrated across two Cadence BioSMB PD multi-column chromatography systems in order to process a 25 L volume of harvested cell culture fluid (HCCF) in less than 12 h. Transfer from batch to continuous resulted in an increase in productivity of the Protein A step from 13 to 50g/L/h and of the MMCEX step from 10 to 60g/L/h with no impact on the purification process performance in term of contaminant removal (4.5 log reduction of host cell proteins, 50% reduction in soluble product aggregates) and overall chromatography process yield of recovery (75%). The increase in productivity, combined with continuous operation, reduced the resin volume required for Protein A and MMCEX chromatography by more than 95% compared to batch. The volume of AEX membrane required for flow through operation was reduced by 74%. Moreover, the continuous process required 44% less buffer than an equivalent batch process. This significant reduction in consumables enables cost-effective, disposable, single-use manufacturing. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

CrossMark

Article history:

Received 29 August 2016

Received in revised form 2 December 2016

Accepted 2 December 2016

Available online 6 December 2016

Keywords:

Continuous chromatography Monoclonal antibody (mAb) Process intensification Productivity Cadence BioSMB PD

1. Introduction

Over 50mAbs have been approved by the FDA and another 50 are currently in phase three clinical trials (Reichert, 2016). The tremendous success of this class of drugs does come at a price, with mAb sales recently estimated to be close to $75 billion (Ecker et al., 2015). However, manufacturing costs are estimated to only be a small fraction of the sales price (Kelley, 2009). This has traditionally led pharmaceutical industries to focus on time to market and adopt a conservative approach to manufacturing, (Castilho, 2014; Gottschalk, 2013; Warikoo et al., 2012). With continuous processing only being employed to reduce the overall purification time for protein therapies whose stability throughout the purification process is limiting (Vogel et al., 2012).

However, the biopharmaceutical landscape is changing rapidly. Growing competition from biosimilars and multiple therapies tar-

geting the same disease have renewed focus upon manufacturing cost (Konstantinov and Cooney, 2015). Many current mAb or Fc fusion protein therapies on the market are facing patent expiration over the next few years (Strohl et al., 2012), enabling manufacturers of biosimilars greater access to market share. Along with cost, flexibility in manufacturing is also critical as the dynamic landscape makes it difficult to predict the quantities of drug required throughout product lifetime and it takes many years to build a new mAb manufacturing facility (Kamarck, 2006).

To address these combined challenges of cost and flexibility, many biopharmaceutical companies are pursuing process intensification via continuous manufacturing (Horowitz, 2010). This endeavor has been actively encouraged by the FDA, which identifies continuous manufacturing as an opportunity to increase the flexibility, agility and robustness of production (Lee et al., 2015).

Process intensification through continuous manufacturing enables the elimination of hold steps which are not value added and further simplifies the process (Warikoo et al., 2012). It also reduces process foot print using smaller and single use equipment which is

* Corresponding author. E-mail address: Mark_Schofield@pall.com (M. Schofield).

http://dx.doi.org/10.1016/jjbiotec.2016.12.005

0168-1656/© 2016 The Authors. Published by Elsevier B.V. This is an open access article underthe CC BY license (http://creativecommons.org/licenses/by/4.0/).

fundamental to flexible manufacturing and the accommodation of dynamic market demand (Fromison, 2009; Hodge, 2004).

A number of companies are now highlighting their continuous purification strategies (Brower et al., 2014; Godawat et al., 2012; Hernandez, 2015; Kaltenbrunner et al., 2016; Mahajan et al, 2012; Pollock et al., 2013; Palmer, 2015; Vogel et al., 2012), but what is largely absent from the literature is how to transfer a batch process to continuous operation (Girard et al., 2015). Here we take a batch mAb purification and describe the experiments and rationale required to transfer it to a continuous chromatography process that can be operated with two Cadence BioSMB PD systems. We show it is possible to achieve higher loading capacities with shorter residence times by loading two or more columns in series. This increase in capacity results from overloading the first column and capturing unbound product in the flow through effluent on subsequent columns. As buffer consumption is inversely proportional to capacity, the increased capacity offered by multi column chro-matography results in significantly reduced buffer consumption. Along with increased capacity, residence times are reduced and so is the cycle time. Increased capacity and decreased cycle both lead to higher process step productivity expressed in gram of product produced per liter of chromatography media and per hour (g/L/h). This can considerably reduce the resin volume required for a purification process. Furthermore, by leveraging the increased capacity that is offered by continuous chromatography, the loading conditions going from batch to continuous can be modified, increasing the robustness of purification.

2. Materials and methods

2.1. Analytical methods

Host cell protein (HCP) was quantified using 3G CHO ELISA (Cygnus Technologies, Southport, NC) assay. Soluble aggregates were measured by HPLC-SEC using a TSK-GEL, Super SW3000, 4.6 mm x 30 cm, 4 |im column (Tosoh Haas, King of Prussia, PA). Concentration of mAb in HCCF was measured using Protein A biosensors (ForteBio, Menlo Park, CA) and an Octet Red 96 BLI system. Concentration of mAb in post Protein A samples was measured using a NanoDrop 8000 spectrophotometer (Thermo Scientific, Waltham, MA).

2.2. Impact of operating parameters on purity of protein A elution

A five factor, two level DoE was designed using Minitab to determine the impact of overloading (loading the columns to saturation) on% recovery of mAb, % mAb aggregate, elution volume and host cell protein (HCP) concentration. The factors tested included residence time (0.6 min vs. 4.5 min), mass loaded (10% DBC- dynamic binding capacity- vs. 90% DBC), load concentration (2 mg/mL vs. 5 mg/mL), and wash volume (10 column volumes (CV) versus 30 CV).

2.3. Capacity determination for multi-column bind/elute processes

HCCF containing mAb at titer 2, 6, and 10 mg/mL was loaded at residence times of 0.5, 1,1.5, 2, 3, and 4.5 min onto KANEKA KanCapA 1 mL Protein A PRC columns (Pall Life Sciences, Port Washington, NY) using an AKTA Avant 25 (GE Healthcare, Uppsala, SWE). The breakthrough curves generated from these experiments were used to calculate the 10% DBC. Operating binding capacity (OBC) for continuous mode was calculated by the method described in Gjoka et al. (2015).

Protein A purified mAb (8mg/mL concentration) titrated to pH 8.2 and 8 mS/cm was loaded at residence times of 0.75,1.5, 2.25, 3, 4.5, and 6 min onto CMM HyperCel MMCEX1 mL PRC columns (Pall

Life Sciences, Port Washington, NY). Tests were repeated using four different load conditions, pH 8 & 6 mS/cm, pH 8 & 10 mS/cm, pH 8.4 & 6 mS/cm, and pH 8.4 & 10 mS/cm at residence times of 1.5 and 3 min. The breakthrough curves were used to measure the 10% DBC at each residence time. Operating binding capacity was calculated using the method described in Gjoka et al. (2015).

2.4. Screening conditions for operation of Mustang Qmembrane and CMM HyperCel sorbent

The impact of pH and conductivity on binding and separation of the target mAb versus host cell protein and aggregate contaminants was determined using a DoE based approach with response surface analysis via Minitab. The AEX membrane, Mustang Q(Pall Life Sciences, Port Washington, NY) and MMCEX sorbent, CMM HyperCel (Pall Life Sciences, Port Washington, NY), were evaluated for binding over a broad range of conductivity and pH. CMM Hyper-Cel sorbent was transferred into an AcroprepTM Advance 96 Filter Plate (PN 8129, Pall Life Sciences, Port Washington, NY). A post Protein A mAb sample at 1 mg/mL in the appropriate buffer was added to each well (Pezzini et al., 2011; Toueille et al., 2011). Filter plates were incubated at room temperature while shaking for 2 h. Flow through was collected and analyzed for recovery of mAb and level of contaminants. For Mustang Qmembrane screening of flow through operating conditions, AcroPrep™ Advance 96-Well Filter Plates with Mustang Qmembrane (PN 8171, Pall Life Sciences, Port Washington, NY) were washed with binding buffer and loaded directly with 1 mg/mL post Protein A mAb samples. Flow through fraction was analyzed for HCP concentration. The best performing conditions from high throughput screening were verified in dynamic mode on 1 mL PRC pre-packed columns (Pall Life Sciences, Port Washington, NY).

2.5. Cadence BioSMB PD chromatographic system

Two Cadence BioSMB PD systems were employed to operate the multi-column chromatography experiments performed in this study. Each system is capable of operating up to 16 columns simultaneously through software that enables the operation of simulated moving bed chromatography. The core technology of the machine is the disposable valve cassette which contains 240 valves and features eight inlets, and six outlets. Valves are activated by air pressure applied by solenoid actuators onto a fluoroelastomer sheet. When air pressure is applied the valves are closed, when the pressure is released pressure in the liquid flow-path forces the valves open. The system is equipped with seven pumps capable of operating from 1 to 200 mL/min. The standard valve cassette flow path is 1 mm in diameter and can accommodate flow rates up to 70 mL/min. A larger 3 mm valve cassette is available for flow rates above 70 mL/min.

The system has four UV sensors with wavelengths in 200-850 nm range, four conductivity sensors with a range of 1 | S/cm to 200 mS/cm, and two pH probes with a range from 2 to 12.

3. Results and discussion

Streamlining of the purification process began by assessing the order of unit operations. The batch process had previously been operated with the sequence of operations, Protein A, MMCEX in bind and elute mode to AEX in flow through mode, Fig. 1. The MMCEX sorbent is eluted via an increase in conductivity, however, the AEX membrane demonstrates improved HCP reduction at low conductivity. To accommodate this, the elution from the MMCEX sorbent has to be diluted three-fold to lower the conductivity from 22 mS/cm to 7 mS/cm. The dilution requires additional buffer and

Protein A

Viral Inactivation

Mixed Mode Cation Exchange

Anion Exchange Membrane

Load to 30 mg/mL at 4 minutes residence

Hold for 30 minutes between pH 3.5 - 3.7

Load to 30 mg/mL Bind at pH 7.6 and 7 mS/cm Elute at pH 7.8 and 22 mS/cm

Dilute and flow through at pH 7.6 and 7 mS/cm

Anion Exchange Membrane

Mixed Mode Cation Exchange

CI ^ I . i_i -j c Load to 30 mg/mL

Flow through at pH 7.6 „. . „,

Bind at pH 7.6 and 7 mS/cm

and 7 mS/cm Elute at pH 7.8 and 22 mS/cm

Fig. 1. Order of unit operations for a batch process.

Table 1

Effect oforder ofunit operations on purity.

Order ofunit operations HCP log reduction % Aggregates

KanCapA - CMM HyperCel - Mustang Q 4.1 1.1

KanCapA - Mustang Q- CMM HyperCel 3.9 1.5

a system for buffer addition and mixing. By modifying the order of unit operations to Protein A followed by AEX and finally MMCEX we can eliminate the dilution step. When we compare the two different sequences of operations we can see an overall similar performance, Table 1. The small advantage in purification performance by operating MMCEX sorbent before AEX membrane is outweighed by the advantages of eliminating the dilution step. This makes integration of the two polishing steps easier and enables the elimination of a system for buffer addition and mixing from the process. Operation without a dilution step also reduces the time required to load AEX membrane as only 1/3rd of the volume has to be applied making the batch process more efficient and less time consuming. This process sequence, Protein A sorbent followed by intermediate purification using an AEX membrane and polishing with MMCEX sorbent, is our starting point as we begin to consider transfer of the process from batch to continuous operation.

3.1. Optimization of protein A capture step

In order to convert the process from batch to continuous operation a DoE was performed on Protein A chromatography media (KANEKA KanCapA). The effects of overloading, wash volume, load concentration and residence time were assessed with regards to the purification performance (data not shown). This experiment shows that a short wash (10 CV divided by 3 wash steps) along with low residence time and overloading has minimal effect on the HCP concentration post Protein A.

To investigate the impact of the multi-column chromatography mode of operation on Protein A binding capacity, multiple single column breakthrough experiments were employed as described in Gjoka et al. With six single column breakthrough experiments, the operating binding capacity in multi-column mode of operation can be approximated over the complete design space. This data presented as a contour plot, using the distance method of interpolation, (Fig. 2) shows a 50% or greater increase in capacity is available in continuous multi-column mode over the range of residence times

that were tested when compared to a single column operated in batch mode.

The design space for column loading capacity was then used to select the optimal binding capacity for a continuous multi-column chromatography process that can accommodate an upstream flow rate of 12 mL/min at a mAb titer of 4 mg/mL which is imposed on the capture step by prior unit operations. When the process was developed only 5 mL pre-packed columns with a 5 cm bed height were available. Applying 12 mL/min onto these columns would give a linear flow rate of 720 cm/h which is in excess of the recommended maximum linear velocity for the Protein A sorbent (500 cm/h). To be able to accommodate this load flow rate, we split the flow across two pairs of columns connected in series, meaning that at any given point in time there are always four columns receiving feed. With this configuration the columns are loaded at 360 cm/h (0.83 min RT per column or 1.66 min total RT as two columns are loaded in series). We can see from Fig. 2 that this gives a 35 mg/mL operating binding capacity. This compares favorably to the batch process which was loaded using a 4 min residence time and a capacity of 27 mg/mL (calculated by taking 60% of the DBC at 10% of protein breaking through).

To make a complete chromatography cycle, four additional columns are required to operate the non-load steps (washes, elu-tion, CIP and re-equilibration). Thus, the Protein A step is operated with eight 5 mL columns (configuration ofcolumns shown in Fig. 7) with a similar overall performance to a predicted four column process with 10 mL columns. This flexibility is a feature that is unique to the Cadence BioSMB PD valve block.

3.2. Low pH viral inactivation

In order to perform post Protein A low pH viral inactivation semi-continuously, a sub-batch cycle to cycle strategy is employed. Eight Protein A elution fractions (the fractions from a complete cycle) are pooled into a first surge tank over the course of one Cadence BioSMB PD Protein A cycle. At the end of the Protein A cycle, this pool is transferred to a second stirred surge tank where acidification to pH 3.6+/-0.1, 30min hold, and a neutralization step with 0.5 M Tris is performed to bring it to pH 8.2 & 6 mS/cm. The neutralized viral inactivated material is then transferred into third surge tank before the end of the second Cadence BioSMB PD Protein A cycle. These steps were repeated for every Protein A cycle using external programmable pumps to transfer product between surge tanks cyclically for the duration of the experiment. Dosing

Residence Time

Fig. 2. Binding capacity of The Protein A sorbent KANEKA KanCapA in (a) Batch mode and (b) Continuous mode with two columns loaded in series.

Fig. 7. Chronograms showing the 3 chromatography processes, (a) Protein A, (b) AEX and (c) MMCEX. Column positions are shown by the radial "spokes" (Column 1, C1, through Column 8, C8). The outer annular ring shows the material the column is receiving and the inner annular ring shows where the flow out of the column is directed. The columns proceed around the cycle in clockwise direction.

was performed using two external pumps that are always running at a fixed flow rate through the Cadence BioSMB PD valve block. The valves within the cassette are used to direct acid and base to the second surge tank or to recirculate the acid and base to their respective containers.

3.3. Evaluation of load conditions - anion exchange membrane adsorber operated in flow through mode

Table 2

Options considered for flow through operation of AEX membrane adsorber.

Strategy # 1 2 3 4 5

Membrane Volume (mL) 10 5 0.86 0.86 0.86

# Cartridges 1 2 2 2 3

Load capacity (g/mL) 2.5 2.5 1.4 2.8 1.4

Regeneration Scheme None None <1 h CIP 1 h CIP 1 h CIP

The key metrics used to assess the performance of the polishing steps are HCP and aggregate reduction. Here an AEX membrane adsorber (Mustang Q) is employed in flow through mode to lower the HCP concentration. The ability of this membrane to remove HCP over a broad design space was investigated using a combination of 96 well membrane plates and a 0.86 mL membrane capsule. First, the HCP removal performance was evaluated at different conductivity and pH load conditions using a fixed load capacity of 0.5 g/mL of membrane. Fig. 3 shows fold reduction in HCP after passing Protein A purified mAb through an AEX membrane under a range of conditions from pH 7.2-8.4 and conductivity from 5 to 10mS/cm. At conductivities ranging from 5 mS/cm to 7 mS/cm and pH 8 to pH 8.4, it is possible to achieve a greater than 60 fold reduction in HCP. To determine the load capacity of the AEX membrane, up to 5 g of mAb was loaded onto a 0.86 mL capsule at four conditions, pH 8 & 5 mS/cm, pH 8 & 7 mS/cm, pH 8.4 & 5 mS/cm and pH 8.4 & 7 mS/cm. These experiments (data not shown) indicate that within this design space, loading Protein A purified mAb beyond 2.5 g/mL of membrane results in an exponential increase in pressure across

the device. This pressure increase occurs before breakthrough of HCP.

With this load capacity limitation, multiple strategies listed in Table 2 that could accommodate a continuous load onto an AEX membrane were considered. An oversized device (a 10 mL capsule) could be employed that is large enough to polish the complete batch without being regenerated and recycled (Table 2, strategy 1). Considering the total load volume to be processed is 25 L of HCCF with a mAb titer of 1 mg/mL, and the AEX membrane loading capacity defined in this study is 2.5 g/mL of membrane, this first strategy could be used to accommodate the load. Alternatively, two AEX 5 mL capsule configured in parallel could be loaded and discarded after use (Table 2, strategy 2). Strategies 3-5 rely on cycling and regenerating a 0.86 mL capsule which can be operated at flow rates up to 10 membrane volumes per minute. These options are feasible since the average flow rate directly post viral inactivation is less than 5 mL/min.The number of devices required depends on two factors: the load capacity (2.5 g/mL) and the amount of time required to regenerate the device (1 h). Here it would be physically possible

Contour Plot of Fold reduction in HCP vs mS/cm, pH

u l/l E

reduction in

m < 10.0

u 10.0 - 20.0

u 20.0 - 30.0

u 30.0 - 40.0

u 40.0 - 50.0

u 50.0 - 60.0

u > 60.0

Fig. 3. AEX membrane, Mustang Q, HCP reduction performance.

to operate with two 0.86 mL capsules and have one device being loaded while the other device is being washed, regenerated and re-equilibrated. Strategies 3 and 4 in Table 2 are both options that use 2 smaller capsules for purification. Using two capsules makes integration with the mixed-mode resin difficult because the cycle time for the membrane adsorbers has to be synchronized with the cycle times for the mixed-mode resin. This means the AEX membrane adsorber cycle time has to be a multiple of the MMCEX cycle time. In strategy 3, one AEX membrane adsorber cycle is exactly twice as long as the cycle for the MMCEX process. This strategy is feasible but requires the CIP step to be shortened to less than 1 h which increases the possibility of performance loss over time. The other option is to create a method with a cycle duration equal to three times that of the mixed-mode cycle which enables us to perform CIP for more than an hour but requires each capsule to be loaded to 2.8g/mL and increases the risk of failure. Therefore, the fifth strategy outlined in Table 2, using 3 capsules each loaded to 1.4 g/mL, was selected because it provided the best compromise in terms of minimizing membrane volume, ensuring the capsules are regenerated thoroughly, and avoiding operation at high pressure. This strategy (5) embraces the concept of continuous processing, reducing the size of consumables by performing more cycles. This also gives a clear path for how best to operate processes over a longer time that would be required to integrate with, for example, a continuous upstream perfusion cell culture process.

3.4. Evaluation of load and elution conditions - CEX mixed-mode resin operated in bind and elute mode

A MMCEX sorbent (CMM HyperCel) is employed in the process to reduce mAb aggregates and to further reduce HCP concentration. Originally, for the batch process a compromise was made between capacity and HCP reduction, Fig. 4. Loading at higher pH results in greater HCP reduction at the binding step, but this is at the expense of capacity. The batch process was designed around column volume. The strategy was to purify the complete batch (25 L HCCF at 1 g/L mAb titer) via a single cycle with a column of 1 L in volume or less. To accommodate this, loading conditions selected for the batch process are pH 7.6 and 7 mS/cm conductivity. The DBC at

10% breakthrough under these conditions is 45 mg/mL. This allows for an operating binding capacity of 27 mg/mL (60% of the DBC at 10% breakthrough).

In continuous mode a higher capacity is expected because columns are loaded in series. This allows the columns to be overloaded without loss of product as product is captured on a subsequent column. Therefore, the loading conditions can be modified when transferring from a batch to continuous process. To find the best compromise between HCP reduction and operating binding capacity a column based DoE was performed using extended pH conditions, Fig. 4. This DoE enables the identification a new optimum design space. Using loading conditions of pH 8.1 and 6 mS/cm conductivity a 45 mg/mL capacity at 1% breakthrough is calculated. A 40 mg/mL operating loading capacity was used for the actual MMCEX process to take a modest margin. These buffer conditions used in continuous multi-column mode decrease the HCP level in elution to 500 ppm compared to 625 ppm obtained in batch mode. In addition, making the same modification to the loading conditions onto the membrane adsorber (upstream of MMCEX) results in increased HCP reduction from the membrane adsorber (60 fold HCP reduction, compared to 30 fold with the batch conditions, Fig. 3). Column elution DoEs were performed to determine the optimal elution condition from MMCEX for yield, HCP reduction and removal of mAb aggregates, data not shown. From these experiments elu-tion conditions of pH 7.8 and 22 mS/cm conductivity are selected to deliver greater than 80% product recovery, >10 fold HCP reduction and a final aggregate clearance of 50%.

With this information, along with the process constraints from the previous process, the MMCEX process can be defined within the context of continuous operation. To generate a balanced cycle, six x 5 mL columns are required in total, where two columns are loaded in series with a residence time of 1.5 min and four columns are required to operate the non-load steps.

3.5. Implementation of the continuous integrated chromatography platform

Two Cadence BioSMB PD systems were connected to operate the entire chromatography train. The first Cadence BioSMB PD system

pH 8.0 8.1 8.2 8.3 8.4

Fig. 4. Extension of MMCEX (CMM HyperCel) sorbent design space made possible by continuous operation.

Process #1

post KanCapA -

post VI/depth/sterile post CMM

300 420 Time (min)

Process #2

post KanCapA

post VI/depth/sterile

post CMM

120 240

Time (min)

Host Cell Protein

80 70 Î60

260 360 Time (min)

Aggregates

260 360

Time (min)

Fig. 5. (a) mAb concentration versus time during continuous operation for process #1. (b) mAb concentration versus time during continuous operation for process #2. (c) Residual HCP levels after polishing steps (d) Residual aggregate levels after polishing steps.

Process #2

Process #1

Process #2

Process #1

performed the capture step with eight x 5 mL Protein A columns along with the dosing for the low pH viral inactivation step. The second Cadence BioSMB PD operates the combined polishing steps. AEX membrane is operated with three devices with a 120 min cycle time. The MMCEX sorbent cycle is 40 min and requires six columns. These two polishing operations are operated on a single Cadence BioSMB PD system with a single pump used to flow-through AEX membrane and load onto two MMCEX columns in series. To accommodate this on a single Cadence BioSMB PD, three MMCEX sorbent cycles are performed per AEX membrane cycle. This again shows the flexibility and intensification offered by this approach. Unit operations are combined even though they have different number of columns and cycle times.

Two continuous integrated processes (process 1 and process 2 in Fig. 5) have been performed to test the performance of continuous processing with this configuration. For both processes, prior to capture step, 20 Liters of clarified CHO supernatant were concentrated using an in line concentrator down to 5 Liters and a final mAb concentration of 4 mg/mL. Each process was operated for a duration of around 10 h. Each Protein A and MMCEX column was cycled 15 times, while AEX membrane capsules were cycled 5 times over the course of each experiment. The mAb production rate, or throughput, was approximately 2 g/h. Sample aliquots were taken every hour for analysis of critical quality attributes. Aggregate and HCP concentration after the final polishing step are shown in Fig. 5. Overall, the continuous purification train resulted in a 4.5

Fig. 6. Impact of continuous processing on consumables.

log reduction in HCP (from 500,000 ppm to 10ppm), a 50% reduction in aggregates (from 2 to 3% aggregates in the load down to 1-1.5% after an isocratic elution from the mixed-mode resin) and 75% total yield. There was no significant time dependent performance observed in the quality of the final polished mAb. The final HCP concentration was approximately five-fold lower compared to the batch process confirming that modifying the design space when transferring from batch to continuous processing can be used to enhance purification performance. The aggregate clearance was 50% for both batch and continuous operation. Notably, the volume ofthe surge tanks between steps was lowered in process 2 resulting in further reduction of the overall process time.

Fig. 6 summarizes the benefits of processing a 25 L HCCF sample volume in continuous processing mode compared to batch mode. Both of these processes assume the same sequence of unit operations being performed under optimized conditions for each respective mode of operation. For the batch mode of operation, it is assumed that the columns are ideally sized to perform the purification in a single cycle. Under these assumptions, the overall process time from start to finish is similar for both continuous and batch modes of operation. However, the amount of sorbent required to operate the continuous process is considerably reduced. For the Protein A sorbent, continuous operation results in a 94% reduction of resin volume. For the polishing steps, total AEX membrane volume would be reduced by 74%, while the MMCEX resin volume would be reduced by 96%. This is a significant reduction in the amount of resin required and could result in considerable cost savings. Buffer consumption would also be reduced by 44% by transferring from batch to integrated continuous processing. The buffer consumption reduction comes from operating with increased capacity, combining unit operations and intensification of the process through both changing the order of unit operations and reducing the number of column volumes required for some of the process steps. The dramatic reduction in buffer consumption might be a reason to convert facilities that are limited by buffer production to switch to continuous processing. These results show a compelling case for continuous production. However, an overall cost benefit analysis between batch and continuous for various scales of production will be addressed in a subsequent publication.

4. Conclusion

Four mAb purification unit operations, Protein A, low pH viral inactivation, AEX, and MMCEX were optimized for batch processing and converted into continuous unit operations using two Cadence BioSMB PD continuous purification systems. The study

demonstrates that significant operational efficiency and quality advantages can be realized by moving from single column batch processing to multi-column processing on a Cadence BioSMB PD system. The study also demonstrates that further operational efficiency is made possible by connecting and operating multiple unit operations together and those continuous processes can be operated at steady state in order to produce a homogeneous, high purity drug substance from start to finish.

In this study, processing continuously enabled a significant reduction in consumable needs (volume of chromatography media, buffer volume). With such a drastic reduction, it follows that continuous processing will have a proportionate impact on facility footprint leading to a reduction in both operating and capital expenditure. Reduction in capital expenditure could prove valuable when advancing drugs through clinical trials because only a fraction of drugs make it through the FDA. Reducing the capital expenditure for each potential treatment during clinical trials reduces the cost associated with the high risks of failure during clinical trials. Furthermore, this type of process intensification, where multiple columns are cycled far more frequently than in batch processing, paves the way for dynamic process control using multi-variate data analysis in combination with several in-line process analytical technologies. This means that in the near future, real-time release of drug product will be possible. This will reduce the need for large inventories of a given drug substance and ultimately lower the cost of that drug to the patient.

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