Although the concept of single use is not new, and has become standard practice for products such as filter capsules and storage bags, today’s single-use processes are much more complex than conventional production strategies and comprise an integrated model—particularly in new facilities—that often involve the replacement of stainless steel equipment.

Single-use technology is already understood to reduce the danger of cross-contamination, decrease investment risk, offer high levels of flexibility and provide cost-effective drug development options for investigational products. Considering that, for example, single-use bioreactors didn’t exist 20 years ago, the technology has gone from evolution to revolution in less than two decades.

More recently, as a result of optimized strains and improved culture media in the field of monoclonal antibody (mAb) production, the productivity of fed-batch mammalian cell culture systems has increased significantly. Biomass concentrations have increased from 1–3 g/L to 7–10 g/L, which facilitates the use of smaller-scale production vessels and, at the same time, also poses a downstream challenge.^1,2 Higher, more concentrated yields are difficult to process with depth filters. For volumes greater than 500 L, the use of centrifuges has become essential to reduce both production costs and waste. However, the introduction of a ready-to-use clarification system that enables single-use processing at volumes of up to 2,000 L may provide a commercially viable solution that replaces both centrifuges and depth filters in a single step.

Harvesting Technologies
High product titers derived from increased cell densities, as opposed to increased specific productivities per cell, result in a considerable solid mass that challenges commonly used harvesting techniques. Currently the most widely applied single use harvesting technology is depth filtration. However, depth filters tend to block at lower loading capacities with higher biomass concentrations.

Higher contaminant concentrations also make depth filters more sensitive to batch variation, which can lead to an oversizing of the filter area (up to 50%) to compensate for fluctuating filtration capacities. Single-use cell removal solutions such as centrifugation are available, but as yet lack capacity, which drives up costs and increases waste.

A solution is, however, available. Sartoclear Dynamics from Sartorius Stedim Biotech is a new single use technology for harvesting animal cell cultures with high cell densities. Offering consistent results, ease of use, speed and flexible scalability, this ready-to-use clarification system—inspired by the blood plasma fractionation industry—is based on the principles of body feed filtration (BFF). Able to process capacities of up 2,000 L, it can replace both centrifuges and depth filters in a single step. The key factor is the addition of a highly porous filter-aid to the feed stream, which increases the permeability of the filter cake and prevents the filter from becoming blocked.

Body Feed Filtration
Body feed filtration uses filter-aids such as diatomaceous earth (DE) to increase filter capacities, which has a long history of use.³ DE, diatomite or kieselgur/kieselguhr, is a naturally occurring, soft, siliceous sedimentary rock with a typical particle size 10–200 µm. Composed of the shells or exoskeletons of fossilized diatoms (microscopic, single-cell algae) and benefiting from a very porous structure and inert character, DE is often used in the biotechnology industry in depth filters during the harvesting stage. DE is added to the cell culture broth where it plays a dual role: it sieves out sub-micron particles and creates a permeable filter cake that improves filter throughput. The minute filter-aid particles provide countless microscopic channels that entrap suspended impurities but allow clear liquid to pass through without clogging.

How BFF Works
A number of products are now manufactured using BFF-based systems, including intravenous immunoglobulin (IVIG), albumin and certain clotting factors, that are based on the principles of selective pH-induced precipitation, ionic strength, the addition of alcohol and temperature shifts.4–6 The precipitates are removed by DE-enhanced depth filtration.

In practice, when the solid concentration becomes too high in biological process fluids that need clarification, the filter cake on the surface of a filter becomes impermeable (Figure 1). Adding a filter-aid, such as DE, creates a more permeable filter cake, which prevents blockage. The amount of DE required depends on the particle concentration. To achieve ideal results and to use as little DE as possible, pre-conditioning the cell culture fluid is recommended. Flocculants can be added or the pH can be lowered, which will create aggregates that are too big to migrate into the filter cake. As such, the cake stays permeable and the DE: biomass ratio can be reduced to about 1:4.

And, although a wide range of DE grades, which differ in purity and permeability, are available for different applications, Sartorius Stedim Biotech selected Celpure C300 as the best filtration aid for mammalian cell culture procedures. Designed specifically for pharmaceutical applications and produced using a patented manufacturing process, Celpure C300 is a highly purified form of DE that offers improved operational consistency. For a variety of tested cultures, media and viabilities, the optimum DE concentration was found to be 40–50% of the wet cell weight (WCW), which could be reduced to 20–30% if the pH was lowered to 5.

Benefits of Low-pH Precipitation
Performance differences in cell removal technologies such as microfiltration and depth filtration can be attributed to the precipitation of smaller particles at low pH levels (pH 4.3–5.5).^4,5 Cell debris, DNA and host-cell proteins all become less soluble in acidic conditions.6 Plus, lowering the pH leads to the formation of larger particles and makes the submicron particles present at pH 7.0 completely disappear (Figure 2). Similarly, BFF also benefits from lower pH conditions: tested cell culture supernatants in acidic conditions showed much clearer filtrates than their neutral counterparts (data not shown).

It is thought that large particles (>0.4 μm) become entrapped in the DE, lowering its overall permeability and filtration capacity. By increasing the DE concentration, it is possible to improve its capacity to retain small particles without losing the required cake permeability. And, although most antibodies are stable at acidic conditions, some are likely to co-precipitate.^5,6 In laboratory scale BFF tests at low pH values, recovery rates of greater than 85% were achieved (data not shown).

Practical Application
As BFF has become more frequently used as a substitute cell culture harvesting solution, Sartorius Stedim Biotech, together with Rentschler Biotechnologie GmbH, attempted to evaluate the technology as a potential single-use alternative to centrifuges and depth filters.⁷ Optimized conditions in a 600 L cell culture production process were tested to assess the scalability of BFF technology. Using Sartoclear Dynamics products, which are designed to be used for the clarification of mammalian cell cultures in pharmaceutical production processes, the results obtained from a number of cell lines and culture media are described.

Pilot-scale production tests were done using a high cell density Chinese hamster ovary (CHO) cell fed-batch culture (17.6 x 106 cells/mL, 95% viability, 1000 L).⁸ The largest BFF test involved 600 L at pH 5.0 (WCW = 8%). During the scale-up experiment, seven process-scale modules with a total filtration area of 1.61 m² in a universal stainless steel holder were used.

Comprising two polyethylene filter plates, the filter cassettes retain the DE and biomass. Cellpure C300 DE (12 kg) was added to the 600 L bulk harvest and pre-filled DE bags were connected with a dust-free adapter to a mixing bag for fast and safe DE transfer. After 5 minutes of gentle mixing, the DE powder had dissolved in the cell suspension; and, prior to filtration, the pH of the resulting mixture was adjusted to 5.0 and mixed for 2 h at 140 rpm.

Figure 3 shows the pressure increase during the process. Filtration was terminated when a pressure value of 1.3 bar was reached and the crude harvest had been filtered. A high and stable flux of approximately 300 L/m2/h was maintained throughout and, overall, a capacity of 311 L/m² was achieved. Low turbidity levels (5–8 NTU) were recorded in the clarified harvest stream during filtration. Following neutralization, the final pool exhibited a turbidity of 41 NTU, which was considerably higher, possibly due to inadequate dosing of the neutralization buffer.

In a small-scale parallel test, improving the neutralization step prevented the turbidity build up: an integrated ready-to-use process skid is under development that will enable controlled inline pH adjustment. For example, IgG1 recovery was recorded at 85% (Figure 3) and, in the future, an optimised neutralization procedure and enhanced post-filtration flushing should further improve mAb recovery. Contaminants such as a host-cell protein and DNA were monitored throughout the process and, in the final pool (after buffer flush and neutralization), those levels were reduced from 841 to 629 mg/mL and 13.8 to 5.0 μg/mL, respectively.

Results
Pilot-scale BFF experiments confirmed previously observed laboratory based findings: reducing the pH to 5.0 after adding DE to the crude cell-culture supernatant gives the best results in terms of filtration capacity, flux and contaminant removal. In addition, using only seven filtration modules, a 600 L harvest was processed in 1 h. As a module holder can accommodate up to 33 modules, it is estimated that a harvest volume of 3,000 L could be filtered in the same time. As such, this method facilitates the effective clarification of crude, high-density cell culture harvests in a single-use large-scale setup.

Conclusion
Wishing to demonstrate the universal application of a novel single-use harvest method for high density mammalian cell cultures, tests using crude harvests from different cell lines and culture conditions enabled the optimal DE concentration as a filter-aid to be determined. In addition, at low-pH levels, a 50% reduction in the amount of filter-aid required was identified.

A robust and reliable single-use clarification system that eliminates the use centrifuges and provides consistent results, ease of application, speed and linear scalability, when combined with precipitation, effectively converts the cell harvesting step from a two-stage process into a single-stage operation, saving both time and money.

Scalable technology such as BFF will close one of the biggest gaps in the single-use product market. With 2000 L now established as the standard size for single-use bioreactors, the elimination of centrifugation to remove cells from such volumes is a huge step forward for the biopharmaceutical industry, enabling a fully single-use process, which brings enormous flexibility and significantly reduces capital investment. Specially designed for cGMP processing, Sartoclear Dynamics consists of prefilled single-use bags containing ultrapure diatomaceous earth in a choice of 0.5–10 kg. With a new quick-connect adapter for dust-free powder transfer, DE can be directly mixed into the cell culture fluid. This porous filter aid prevents filter blockage and, as the system maintains a constant ratio of biomass and filter-aid, users will benefit from continuous and optimized filter performance. Able to clarify even very dense crude cell harvests at high flow rates, BFF makes economic and productivity sense. 

References

1. B. Kelley, “Industrialization of MAb Production Technology: The Bioprocessing Industry at a Crossroads,” MAbs 1(5), 443–452 (2009).
2. M. Buttler, “Animal Cell Cultures: Recent Achievements and Perspectives in the Production of Biopharmaceuticals,” Appl. Microbiol. Biotechnol. 68(3), 283–291 (2005).
3. E.J. Cohn, et al., “Preparation and Properties of Serum and Plasmaproteins, IV: A System for Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissue and Fluids,” J. Am. Chem. Soc. 68, 459–475 (1946).
4. J. Curling, et al., Production of Plasma Proteins for Therapeutic Use (Wiley-Blackwell, Hoboken, New Jersey, USA, 2012).
5. J. More, et al., “Purification of Albumin from Plasma,” in J. Harris (Ed.), Blood Separation and Plasma Fractionation (Wiley-Liss, Hoboken, New Jersey, USA, 1991).
6. M. Stucki, et al., “Investigations of Prion and Virus Safety of a New Liquid IVIG Product,” Biologicals 36, 239–247 (2008).
7. M. Westoby, et al., “Effects of Solution Environment on Mammalian Cell Fermentation Broth Properties,” Biotechnol. Bioeng. 108(1), 50–58 (2011).
8. M. Trexler-Schmidt, et al., “Identification and Prevention of Antibody Disulfide Bond Reduction During Cell Culture Manufacturing,” Biotechnol. Bioeng. 106(3), 452–461 (2010).