Scale-Up of Multi-Column Chromatography Using the Cadence™ BioSMB Process System

Pall’s Cadence BioSMB platform is the first disposable flow path,continuous multi-column chromatography solution that is fully scalable from the Process Development (PD) laboratory to GMP Manufacturing. The Cadence BioSMB Process system is specifically designed to allow users to convert an existing PD scale continuous purification into a process scale continuous purification based on feedstreams derived from fed batch bioreactors of up to 2000 L. The chromatographic process developed using the PD system can be readily scaled to the Process system without changing the sorbent, buffer system or product quality assays. BioSMB technology provides process consistency across all scales of operation while making it practical to implement in flexible, multi-product facilities.

• Inherent unit operation efficiency of the BioSMB technology reduces the cost of chromatographic media by up to 80% without changing either the sorbent or buffer used in the chromatographic process
• Entire chromatographic process can be conducted in a smaller system using smaller column volumes with associated reductions in buffer that would be required in a standard batch process to produce the same amount of material
• The entire single-use flow path of the Cadence BioSMB Process system can be replaced within 30 minutes
• The valve system does not require cleaning or cleaning validation
• Ability to use up to 8 columns increases specific productivity and application flexibility

Scale-Up Study
The scale-up of a monoclonal antibody (mAb) capture process was performed using the Cadence BioSMB platform. A humanized IgG1 was expressed in a CHO-K1 cell line using a 500L Single-Use Bioreactor (SUB). The harvested cell culture fluid (HCCF) with a titer of 5.8 g/L of expressed mAb was purified using KANEKA KanCapA* Protein A sorbent. Method development was carried out initially using batch chromatography and following optimization of the chromatographic conditions was transferred to multi-column operation using the PD system. The continuous process was then scaled-up 150-fold via the Process system. The entire study was accomplished in less than 3 weeks.

Process development via batch chromatography
The dynamic binding capacity of KANEKA KanCapA sorbent for the mAb was determined to be ~45 g/L at a residence time of 6 minutes. The non-load stages of the purification (i.e., wash, elution, regeneration, re-equilibration) required 22.5 column volumes (CV) to complete.

Cadence BioSMB PD System Study
The optimal multi-column chromatography process was determined to use:

– 1 primary load column
– 2 secondary load columns in parallel (to accommodate the effluent from Wash 1), and
– 2 columns in the non-load steps

Loading was carried out using 8 CV of HCCF over 10 minutes. A total of 5 x 5 mL KANEKA KanCapA PRC columns (1.12 cm i.d. x 5 cm) were used for the capture step operating at a flow rate of 4.0 mL/min, giving a residence time of 3 min 02 sec. Under these conditions, the operating binding capacity of KANEKA KanCapA sorbent for the mAb was 46.4 g/L (Table 1).

 Cadence BioSMB Process System Study
The 5 column process was scaled up 150-fold using a total of 5 x 0.77 L KANEKA KanCapA OPUS* columns (14 cm i.d. x 5 cm) operating at a flow rate of 37 L/h, giving a residence time of 3 min 02 sec and an operating binding capacity for the mAb using KANEKA KanCapA sorbent of 46.4 g/L. Under these conditions, 400 L of HCCF containing 2.3 kg of expressed mAb (5.8 g/L) was processed in 13 cycles over 11 hours (Table 1).

 

Scale-Up Results
Performance data of the Cadence BioSMB PD and Process systems is summarised in Table 2 and compared to the batch benchmark. Product yield and quality was consistent throughout the 150-fold scale-up of the process and comparable to the batch benchmark.
As a consequence of the multi-column loading approach, higher protein binding capacity was achieved and the mAb concentration in the eluent from the KANEKA KanCapA column was increased by 30% compared to the batch procedure. Furthermore, by operating at a lower residence time compared to batch, the multi-column approach generated a 3.5-fold productivity improvement yielding 56 g mAb /L KANEKA KanCapA sorbent /h versus 16 g/L/h for the comparable batch process.
This study represents ~10% of the maximum operational throughput of the Cadence BioSMB Process System.

Process Comparison
The batch process can be modelled in order to compare how it would perform compared to the scale-up study using the Cadence BioSMB Process system. The data are summarised in Table 3. In order to purify mAb from 400 L of HCCF using a batch process under similar processing times, one option is to run a 20 L Protein A chromatography column over 3 cycles, compared to the multi-column process where the optimal process requires just 3.85 L of Protein A sorbent packed into 5 columns each of 0.77L volume, and operated over 13 cycles.
This demonstrates a potential overall saving of 16.15 L Protein A sorbent using the Cadence BioSMB Process system compared to a conventional batch technique.

Summary
• The BioSMB platform offers a scalable path to clinical manufacturing
• A total of 2.3 kg mAb was purified in 11 hours (13 process cycles) using the Cadence BioSMB Process System
– Product yield and quality was consistent with a batch process
• Only 3.85 L KANEKA KanCapA sorbent was required
– A batch process would require ~20 L KANEKA KanCapA sorbent
• 80% reduction in Protein A volume and associated storage, handling, processing and disposal costs
• 5 columns was the optimal configuration
• The batch process was converted to continuous and scaled up all within 3 weeks
• The BioSMB PD process was successfully scaled up >150-fold
– Chromatographic performance was comparable using the Cadence BioSMB PD and Cadence BioSMB Process systems
Product quality attributes were similar throughout scale-up

 

Acknowledgement
Grateful acknowledgement is given to Mark Brower, Nuno Pinto, Doug Richardson, Bhumit Patel, Jun Heo, Sen Xu, Mike Cuzzola and Ed Glowacki of Merck & Co., Inc. for facilitating this scale-up study.

 *KANEKA KanCapA is a trademark of KANEKA Corporation. OPUS is a trademark of Repligen Corporation. ® indicates a trademark registered in the USA and TM indicates a common law trademark.

www.pall.com/biopharm



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