Sterilizing Grade Filtration Unit Operations for Plasmid DNA Processes

The last step in plasmid DNA (pDNA) purification is final sterile filtration. This step ensures the plasmid DNA product is sterile and safe for administration to patients.

• Components of a Sterilizing Grade Filtration Unit Operation
• Sterile Filtration: Parameters to Optimize
• Key Considerations and Challenges to pDNA Filtration
• Plasmid Size Impacts Sterile Filtration Yields
• Buffer Salt Concentration Impacts Filter Capacity and Yields
• Supercoiled pDNA Content Increases Filtration Capacity
• Membrane Material Impacts Capacity and Flux in pDNA Filtration
• Plasmid DNA Concentration May Affect Yield and Capacity
• Plasmid DNA Filtration Parameters Summary
• Related Products

Components of a Sterilizing Grade Filtration Unit Operation

Sterilizing Grade Filtration unit operations for pDNA processes should include:

• A membrane that will remove bacteria and particulates from the feed stream (ex: Millipore Express^® SHC filters)
• A membrane that provides high capacity, high flux and allows excellent pDNA transmission  (eg. Millipore Express^® SHC filters)
• A filtration system that enables efficient sterile filtration, high pDNA recovery, and simplifies integrity testing without compromising sterility of the flow path.

Sterile Filtration: Parameters to Optimize

A sterile filtration unit operation for pDNA downstream processing can be optimized by changing the:

• Filter membrane (i.e. material and pore size)
• Filter format device and filtration system design
• Operating conditions (i.e. flowrate or pressure)
• Filter loading (minimize fouling to maximize yield)
• Formulation buffer chemicals
• pDNA solution purity (minimize host cell impurities)
• pDNA conformation (supercoiled, linear, open-circular, etc.)

Key Considerations and Challenges to pDNA Filtration

The large size of pDNA can present a challenge for sterile filtration unit operations as pDNA transmission can be low, leading to yield loss and low filter capacity. In addition, the large molecule size increases viscosity resulting in low flow rates. Finally, the presence of adjuvants, which are often used to enhance immunogenic responses to DNA vaccine products, can reduce bacterial retention with sterilizing-grade membrane filters. Key considerations are summarized in Table 1.

Table 1. Key considerations for sterile filtration of pDNA solutions.

Attributes	Parameters	pDNA Product Characteristics
Sterility assurance	Membrane pore size	Large size of pDNA
Particulate reduction	Membrane chemistry	Viscosity of pDNA solution
Filtration capacity and flux	Operating conditions (flowrate or pressure)	Validating bacterial retention for adjuvanted pDNA solutions
pDNA yield	Formulation chemicals
	Filter loading

Plasmid Size Impacts Sterile Filtration Yields

Process parameters of the sterile filtration step should be optimized to maximize the filter capacity and pDNA transmission or yield. Our studies have shown that the capacity, flux, and yield of pDNA feed streams during sterile filtration varies significantly, and is correlated to plasmid size.

For plasmids less than 10 kbp in size, developing a robust sterile filtration operation can be as simple as confirming filter sizing using Vmax™ or Pmax™ methodology¹. However, as pDNA increases in size from 10 - 20 kbp, or larger, the challenges increase, impacting pDNA transmission and overall process yield^2,3. Table 2 summarizes sterile filtration performance from different various studies. 

Table 2. Expected performance for sterilizing-grade filtration of purified pDNA based on internal studies and literature search.

Plasmid DNA Size (kbp)	Expected Sterilizing Grade Filtration Yield (%)	Expected Sterilizing Grade Filtration Capacity (L/m2)
<10	>90	>50
10-20	>80	Variable
>20	<80	<20

Buffer Salt Concentration Impacts Filter Capacity and Yields

While pDNA size impacts sterilizing filter performance, internal data, and published studies show that buffer composition can also affect filtration performance. 

Reducing the salt concentration in formulation buffer from 150 mM to 20 mM resulted in a threefold increase in sterilizing-grade filter capacity for a 6.4 kbp supercoiled plasmid DNA². It was speculated that this effect was the result of salt concentration directly affecting the pDNA radius of gyration and diffusion coefficient which impacts filterability^4,5,6,7. By contrast, for larger plasmids of 72 and 116 kbp in size, the presence of 150 mM NaCl was shown to improve filterability by increasing transmission through PVDF membranes by 47% and 11% respectively.

Supercoiled pDNA Content Increases Filtration Capacity

Studies have shown that supercoiled pDNA is easier to filter than open-circular pDNA and thus the purity of supercoiled pDNA can significantly impact filtration step efficiency. Even a small percentage of open circular pDNA can quickly foul a sterilizing membrane reducing filter capacity for smaller supercoiled pDNA. One study reported a 10-fold increase in filtration yield by increasing the levels of supercoiled DNA from 90% to 95% ².

Membrane Material Impacts Capacity and Flux in pDNA Filtration

Membrane material affects the capacity and flux of sterilizing filters. Although both polyvinylidene fluoride (PVDF) and polyethersulfone (PES) membranes can be used to successfully filter pDNA solutions, PES membranes are preferred, particularly for larger plasmids, because they improve pDNA transmission and minimize pDNA damage³. Internal studies also confirmed higher pDNA yield using PES asymmetric membrane filters. However, not all PES membranes are equivalent: the pore structure and hydrophobicity of PES membranes from different filter manufacturers can impact filter capacity ².

Plasmid DNA Concentration May Affect Yield and Capacity

Data from internal and published studies suggest that pDNA concentration can affect filtration yield and capacity. Some published reports have shown increased mass throughput with high pDNA concentration². However, results from internal studies suggest a more complex picture: dependent on the buffer and pDNA purity, higher pDNA concentrations may promote self-association of the molecules, resulting in lower filter capacity and yield. While concentration of pDNA is certainly a critical operating parameter, specific approaches for optimizing performance via dilution or concentration should be optimized for each pDNA template and purification process.

A review of sterile filtration operating conditions showed that operating flux or pressure has little to no impact on filtration capacity or yield. However, for larger plasmids in particular, a higher force could compromise pDNA integrity due to mechanical stress. 

Plasmid DNA Filtration Parameters Summary

Time spent optimizing the operating conditions for pDNA filtration can markedly improve the step efficiency of unit operations. Some features such as plasmid size cannot be adjusted but other parameters such as plasmid purity, buffer composition, and filtration membrane can be optimized to improve filtration efficiency. Ultimately, early optimization will reduce costs and lead to a more sustainable filtration operation.

• Yield and capacity can be optimized by adjusting salt concentration, pDNA purity, pDNA concentration and defining the operating conditions to avoid extreme fouling.
• Implementing PES membrane filters maximizes pDNA transmission and yield.
• Product integrity should be consistently monitored to minimize possible shear damage during the filtration operation. 

We have a broad portfolio of sterilizing membrane filters for your pDNA downstream sterile filtration operation: 

References

1. “Filter Sizing Methods“ Application Note – Lit. No. AN1512EN00 rev – 3/00. Printed in U.S.A. © 2000 Millipore Corporation

2. Watson M, Winters M, Sagar S, Konz J. 2006. Sterilizing Filtration of Plasmid DNA: Effects of Plasmid Concentration, Molecular Weight, and Conformation. Biotechnol. Prog.. 22(2):465-470. https://doi.org/10.1021/bp050280s

3. Kong S, Titchener-Hooker N, Levy MS. 2006. Plasmid DNA processing for gene therapy and vaccination: Studies on the membrane sterilisation filtration step. Journal of Membrane Science. 280(1-2):824-831. https://doi.org/10.1016/j.memsci.2006.03.003

4. Latulippe DR, Zydney AL. Radius of gyration of plasmid DNA isoforms from static light scattering. Biotechnol. Bioeng.. 107(1):134-142. https://doi.org/10.1002/bit.22787

5. Hammermann M, Steinmaier C, Merlitz H, Kapp U, Waldeck W, Chirico G, Langowski J. 1997. Salt effects on the structure and internal dynamics of superhelical DNAs studied by light scattering and Brownian dynamics. Biophysical Journal. 73(5):2674-2687. https://doi.org/10.1016/s0006-3495(97)78296-1

6. Nguyen TH, Elimelech M. 2007. Adsorption of Plasmid DNA to a Natural Organic Matter-Coated Silica Surface:  Kinetics, Conformation, and Reversibility. Langmuir. 23(6):3273-3279. https://doi.org/10.1021/la0622525

7. Latulippe DR, Zydney AL. 2008. Salt-induced changes in plasmid DNA transmission through ultrafiltration membranes. Biotechnol. Bioeng.. 99(2):390-398. https://doi.org/10.1002/bit.21575