三种不同细胞培养工艺成本比较：补料批次、浓缩补料批次和浓缩灌流

An Economic Comparison of Three Cell Culture Techniques: Fed-Batch, Concentrated Fed-Batch, and Concentrated Perfusion

1. Janice A. C. Lim (Bioprocess Consultant) 1. John Bonham Carter (VP Sales)

2. Andrew Sinclair (Managing Director) 2. Jerry Shevitz (President)

Biopharm Services Ltd Refine Technology, LLC

Lancer House, East Street, 26 Chaplin Road, Unit 1107

Chesham HP5 1DG PO Box 691

United Kingdom Pine Brook, NJ 07058, USA

Contact: Andrew Sinclair Contact: John Bonham Carter

Tel: +44 (0) 1494 793 243 Tel: +1 793-993-3003

a.sinclair@https://www.360docs.net/doc/8f633890.html, jbonhamcarter@https://www.360docs.net/doc/8f633890.html,

https://www.360docs.net/doc/8f633890.html, https://www.360docs.net/doc/8f633890.html,

Photo 1: The operator is handling a ATF6 System in the plant.

Table 1: Guideline working volume sizes for each ATF system

selected nutrients during the growth culture cycle to improve productivity and growth. The culture is subsequently harvested and the product recovered. Fed-batch culture has been an attractive choice for large-scale production due to its operational simplicity and familiarity as a carryover process from fermentation. However, fed-batch mode of operation typically also involves high start-up costs, resulting from the need for larger bioreactor plant capacity.

In perfusion culture, a continuous supply of fresh media is fed into the bioreactor while growth-inhibitory by-products are constantly removed. The increasing interest in the use of perfusion culture can be attributed to the higher product output from a reduced reactor size (hence, simplifying operation, cleaning and sterilization). The cell densities achieved in perfusion culture (30–100 x106 cells/mL) are typically higher than for fed-batch modes (5?25 x106 cells/mL) . The principal aspect of perfusion operation, which is different from fed-batch, is the added requirement of a cell retention device. Cell retention systems add a level of complexity to the process, requiring management, control and maintenance for successful operation. Perfusion bioreactors can suffer operational difficulties such as malfunction or failure of the cell separation device which can lead to shortening of the production run, leading further, to increased operating costs. This has previously limited their attractiveness.

In recent years, a new platform technology has been developed for biologicals production. The ATF? System, introduced by Refine

Technology, LLC (Pine Brook, NJ). Used in the alternating tangential flow mode, it is a low shear filtration system that inhibits filter-membrane fouling. This external cell-separation system is able to maintain

continuous culture for extended periods of time and offers the capability of rapid filter change without compromising the culture run.The ATF? System allows increased volumetric productivity and reduced bioreactor size.

Concentrated fed-batch and concentrated perfusion are two production techniques based on the ATF? System, which simultaneously nourishes the culture and concentrates the product within the bioreactor. These manufacturing methods permit great increases in cell and product concentrations as compared to fed-batch and perfusion. For example in the concentrated fed-batch production platform, one of Refine Technology’s pharmaceutical clients4 has reported a protein product titer of 17g/L with an unoptimized CHO cell process. Higher titers are expected as process

optimization continues. In the concentrated fed-batch operation, ultra high cell densities of (70–200)x106 cells/mL have been achieved; similarly, extremely high cell densities in the region of (70–100)x106 cells/mL have been achieved in systems using the concentrated perfusion mode.

The system scales on a linear basis from 1L to greater than 1,000L and can be used with traditional or disposable bioreactors and with all cell types including anchorage dependent lines. Table 1 indicates the working volume sizes for each ATF? System in the scale-up process. The figures in the table are provided as guidelines. Actual capacity and vessel size depend upon process conditions.

This article compares the economic feasibility of a typical glycosylated protein manufactured using three production techniques – fed-batch (FB), concentrated fed-batch (CFB) and concentrated perfusion (CP). The Excel-based process-cost modeling tool, BioSolve from BioPharm Services Ltd (Chesham, Buckinghamshire, UK), was used for the economic assessment. The methodology, assumptions and key results of the cost model are described. The analysis will use the cost of goods (CoG) metric expressed on a per gram basis for comparability.

Evaluation Methodology

The commercial process-cost modeling software, BioSolve, is used

to assess the process economics of the FB, CFB and CP production techniques. The CoG is a useful metric to quantify the operating costs of

a manufacturing option. BioSolve is an Excel-based tool which determines the CoG by accounting for the indirect (fixed) overheads of the facility and the direct (variable) operating costs of the process. The fixed cost element consists of the capital charges, taxes, insurances while the direct costs include the consumables, materials, labour and waste management. Such an approach to estimate the CoG has been used to evaluate the process economics of disposable membrane chromatography for use in biomanufacturing.

Figure 1 illustrates the basic BioSolve framework, which comprises the user interface, process definition, productivity and cost calculations, and model outputs. The interface allows a rapid assessment of the impact of pre-defined key input parameters (e.g. process scale, product titre, disposable options) on the model outputs. The core of the cost model consists of the process definition, which include the mass balances, equipment sizing, operating parameters and resource allocation. The calculated items include the plant productivity, labour requirements, consumable and materials usage, and equipment list. The costs of these components are maintained in a cost database, which is built with data consisting of benchmarking information from several biomanufacturing operations. The key outputs generated by the cost model are the facility throughput, bill of materials, CoG and capital investment. Case Study Background

The model compares the manufacture of a glycosylated protein in three different production techniques – FB, CFB and CP. Figure 2 depicts the process sequences for these production methods. The seed train for the FB process is longer as this option requires more steps to scale-up to the production cell culture bioreactor. The FB upstream process information was derived from commercially-relevant operations commonly practiced in the industry. The upstream information for the CFB and CP processes was provided by Refine Technology, based on data supplied by its clients. In the FB and CFB processes, the removal of cells is achieved by centrifugation and depth filtration. Such recovery unit operations are not required in

the CP process as the cells are retained in the ATF? System. The three processes were assumed to have the same purification sequence. This downstream sequence was selected according to commercial relevance and sourced from details that are available in the public domain. Table 2

summarizes the key operating parameters used in the base case study.

Figure 1: Framework of BioSolve

Figure 2: Process sequence (a) FB, (b) CFB and (c) CP for the production of a

glycosylated protein

For the FB process, all the seed steps included in the model are assumed to be batch fermentations. In the production cell culture step, two additional feeds are added to the bioreactor 3 days and 6 days after inoculation. The final growth phase takes about 13 days.

The CFB and CP seed sequence is performed using a 200mL bag carried out in batch fermentation mode, followed by a 20L bioreactor also run in the CFB mode.

In the FB and CFB processes, the recovery procedure is Centrifugation followed by Depth Filtration. A UF/DF step is used to concentrate the

product stream prior to the Protein A unit operation. A Virus Inactivation step occurs. The product stream is subsequently loaded onto an IEX Bind & Elute column and passed through an IEX Flow Through column. A Viral Filtration step takes place prior to the UF/DF step. A final Sterile Filtration step is used to purify the product. This purification sequence is based on typical unit operations for the production of a glycosylated protein, using typical solutions and timings. The overall yield for the downstream sequence is 50% and 69% for the FB/CFB and CP processes respectively.

a. Scenario 1 – Base Case

Figure 3 capacity, which translates to lower initial investment costs. The CFB and CP technologies offer substantial capital charge reductions of 13% and 32% respectively relative to the FB process. The CoG per gram is inversely proportional to the annual product mass with the CP process producing the most product (265 kg/year) and FB producing the least amount of product (130 kg/year). This correlation between low CoG and high annual product mass is expected, as increased throughput of product will result in lower costs per gram produced.

In the CP process, the materials costs contribute a significant amount to the CoG. This can be attributed to the intensive usage of media over the extended perfusion culture cycle.

The model results indicate that the CP process (scenario 1c) is the most cost effective technology with the lowest overall CoG per gram of $87/g. The CFB process (scenario 1b) is slightly more expensive at $118/g. The FB process (scenario 1a) has the highest CoG ($158/g). The CFB and CP options provide cost savings of about 25% and 45% respectively when compared to the FB process.

Table 1: Guideline working volume sizes for each ATF system

(1) Cell concentration 50x106 cells/ml x 0.2L, diluted to 0.5x106 in 20L (2) Cell concentration 50x106 cells/ml x 20L, diluted to 1x106 in 1000L (3) vvd = volume of fresh medium/working volume of reactor/day.

(4)

Concentrated nutrients/factors are required during the run in the fed-batch process.

Figure 3: CoG results for the base case

b. Scenario 2 – Changing the titre for the CFB process

The expression levels in the CFB process can vary depending on the type of cell line and duration of cell line development. In this section, the effect of changing the product titre for the CFB process is examined while keeping the bioreactor size constant at 1,000L (Figure 4). In the worst-case scenario, the titre is reduced from 17g/L (scenario 1b) to 10g/L (scenario 2a). In the best-case scenario, the titre is increased to 25g/L (scenario 2b).

Reducing the titre, as expected, increases the CoG as the annual throughput is reduced. When the titre is reduced to 10g/L (scenario 2a), the CFB process appears less economical as the CoG ($184/g) is higher

than the FB CoG at $158/g (scenario 1a). On the other hand, when the titre

is increased to 25g/L, the CoG reduces to $87/g. This improvement in the

expression level would make the CFB process much more attractive than the FB process with significant cost savings of 45%.

c. Scenario 3 – Changing the bioreactor size for the CP process

The bioreactor size used for running perfusion culture mode can vary. This section looks at changing the bioreactor size of the CP process while keeping the titre unchanged at 0.8g/L. Two other bioreactor sizes (500L and 750L) are considered in this analysis.

When the bioreactor volume is reduced to 500L (scenario 3a), the CP

process generate about the same annual output (132 kg) as the FB process (130 kg). The CoG of the CP process increases from the base figure of $87/g (scenario 1c) to $133/g (scenario 3a). The CP process at 500L and 0.8g/L is still more economical than the FB process. It should be noted that the simplification of material and people flow provided by reduced bioreactor capacity in the CP facility cannot be calculated easily but may provide the key driver to such technology being increasingly used in biomanufacturing.

Figure 4: CoG results for the FB process and CFB

process at 10g/L, 17g/L and 25g/L (1,000L bioreactor scale)

Figure 5: CoG results for the FB process and CP process using 500L, 750L and 1,000L (0.8g/L titre)

200180160140120100806040200

Others Labour Consumables Materials Capital Charge

806040200

200

180

160

1401201008060

40

20

Others Labour Consumables

Materials

Capital Charge

Conclusions

This article has provided an economic comparison for the production of

a typical glycosylated protein using the FB, CFB and CBP technologies. The CFB and CP processes are based on the ATF? System. The model results revealed that in the base case, CoG savings of 25% and 45%

can be achieved using the CFB (1,000L, 17g/L) and CP (1,000L, 0.8g/L) technologies respectively when compared to the FB process (10,000L,

2g/L). The bulk of the savings is derived from the reduction in capital requirements due to higher productivities, resulting in reduced overall bioreactor capacity in these processes. The use of the CFB and CP technologies would seem a potential approach towards lower upfront capital investment and operating costs. Sensitivity analyses were performed on the product titre in the CFB process and the bioreactor volume in the CP process. The feasibility of the CFB technology reduces at the worst-case product titre (10g/L) reported in current industrial processes. The CP CoG is still lower than the FB CoG when the bioreactor volume is reduced to 500L. In addition to cost considerations, there are other key factors that may impact the selection of an appropriate process for a drug candidate. The existing fed-batch and perfusion processes are well-known but both have limitations. The CFB mode with the ATFTM System is recommended if the product of interest is a single concentrated product stream harvested from the reactor. The CP mode can be used to reduce the size of bioreactor required to produce the same amount of product as in fed-batch or perfusion.

This article has not evaluated the use of the ATF? System during the

cell banking and seed expansion stages in the upstream manufacturing process. Performing perfusion cultures with such a system is able to generate large cell mass for high density freezing to be used for cell banks preparation. High cell density freezing reduces the inoculum expansion time by removing the first seed passages and inoculates a small bioreactor directly from the freezer. This leads to reduced manual handling of culture and improved facility utilisation.

The impact of the use of disposable bioreactors in the cell culture operations has not been captured in this article analysis. Disposable systems are easier to set-up and eliminate cleaning requirements; hence the turnaround time for such single-use technologies is typically shorter as compared to their stainless steel counterparts. The faster turnaround time could result in increased plant throughput and affect the production costs of the facility.About the authors:

Janice Lim is Bioprocess Consultant at Biopharm Services Ltd

e-mail: j.lim@https://www.360docs.net/doc/8f633890.html,

Corresponding authors:

Andrew Sinclair is Managing Director of Biopharm Services Ltd,

Lancer House, East Street, Chesham HP5 1DG, United Kingdom

+44 1494 793243, e-mail: a.sinclair@https://www.360docs.net/doc/8f633890.html,

https://www.360docs.net/doc/8f633890.html,

Jerry Shevitz is President of Refine Technology, LLC,

26 Chaplin Road, Unit 1107, PO Box 691,

Pine Brook, NJ 07058, USA, +1 973 952 0002

e-mail: jshevitz@https://www.360docs.net/doc/8f633890.html,

https://www.360docs.net/doc/8f633890.html,

John Bonham Carter is Vice President of Sales

and Business Development of Refine Technology

LLC, 26 Chaplin Road, Unit 1107, PO Box 691,

Pine Brook, NJ 07058, USA, +1 793-993-3003

e-mail: jbonhamcarter@https://www.360docs.net/doc/8f633890.html,

https://www.360docs.net/doc/8f633890.html,

References:

1. Chu, L.; Robinson, D. K. Industrial Choices for Protein Production by Large-Scale Cell Culture. Curr. Opin. Biotechnol., 2001, 12, 180-187.

2. Carstens, Jason. Perfusion! Jeopardy or the Ultimate Advantage? BioProcess International webinar, September 2009.

3. Furey, J. Scale-up of a Cell Culture Perfusion Process. Genet. Eng. News, 2002, 22(7), 62.

4. Mora, J.; Sinclair, A.; Delmdahl, N.; Gottschalk, U. Disposable Membrane Chromatography Performance Analysis and Economic Cost Model. BioProcess Int’l, June 2006.

5. Lim, J. A. C.; Sinclair, A.; Kim. D. S.; Gottschalk, U. Economic Benefits of Single-Use Membrane Chromatography in Polishing – A Cost of Goods Model, BioProcess Int’l, February 2007.

6. Noe, Wolfgang. Paradigm Changes and Technology Gaps in the Biopharmaceutical Industry. Presentation at BioProcess International Conference & Exhibition. Raleigh, NC, USA. 12-15 October 2009.