Ultra-low cross-linked microgel purification using hollow fiber TFF

Gabrielle Montgomery, Schmid College of Science & Technology
and
Andrew Lyon, Fowler School of Engineering, Chapman University, Orange, CA 92866

Hollow fiber TFF as an alternative to both equilibrium dialysis and centrifugation for ULC microgel purification

During synthesis of ultra-low cross-linked (ULC) microgels, a significant amount of linear polymer is formed (~ 50% by mass) as a byproduct, necessitating further purification. In this article we describe purification of ULC microgels using the lab-scale Minimate™ EVO tangential flow filtration system.

Hollow fiber tangential flow filtration (TFF) proved to be a highly effective method for purifying ULC microgels, successfully removing linear polymer contaminants while preserving particle integrity.

Compared to the traditional method of equilibrium dialysis, hollow fiber TFF was able to significantly improve processing time from weeks to 1.5 days.

Introduction

Microgels are micrometer-sized particles composed of crosslinked polymer chains. Their mechanical softness allows them to swell extensively in solvents, with the most common microgels being composed of water-soluble polymers. We and others have employed microgels in various biomedical applications, such as targeted drug delivery (1), hemostasis (2,3), and tissue engineering (4,5). Our group focuses on a specific class of colloidal particles called ULC microgels composed of two monomers: N-isopropylacrylamide (NIPAm) and acrylic acid (AAc). Unlike conventional microgels, ULC microgels do not use N, N'-methylene bisacrylamide (BIS) as a crosslinker, and instead rely upon rare chain transfer reactions to create a very small amount of network connectivity, resulting in crosslinking percentages estimated to be ~ 0.1 to 0.2 mol% (6).

During ULC microgel synthesis, a significant amount of linear polymer is formed (~ 50% by mass) as a byproduct, necessitating further purification. Traditional purification methods such as equilibrium dialysis and centrifugation have limitations. Centrifugation can cause aggregation and material loss and is typically not scalable in the academic laboratory setting. Dialysis is slow and we have found it to be inefficient when removing higher molecular weight linear polymer impurities from our microgel preparations, resulting in very long (weeks) dialysis times. Here, we describe the use of hollow fiber TFF as a method for particle purification. TFF provides a more efficient and potentially scalable alternative for microgel purification. Our optimization of these methods has focused on reducing membrane clogging by utilizing continuous flow, enabling consistent filtration. Unlike centrifugation, TFF eliminates high shear forces, preventing aggregation and preserving the integrity of soft, swollen microgels. Additionally, TFF can process larger volumes, significantly improving purification speed and scalability.

Methods and materials

Ultra-low cross-linked microgel synthesis

ULC microgels composed of 90% N-isopropylacrylamide (NIPAm) and 10% acrylic acid (AAc) were synthesized at the 4 L scale following an aqueous free radical polymerization protocol (6). All glassware was cleaned and rinsed with 18MΩ water prior to use.

Briefly, 63.804 g NIPAm and 4 mL AAc were combined in a 1000 mL volume of 18MΩ water and fully dissolved by stirring at 450 rpm for approximately 20 min. The dissolved monomers solution was then filtered using a 0.2 µm membrane filter and transferred into a 5000 mL jacketed reaction vessel. The reactor was mechanically stirred at 250 rpm, and the solution was heated to 70°C. While the reaction mixture was heating, an additional 2900 mL of filtered 18MΩ water was transferred to the reaction vessel. The reaction mixture was blanketed under a flow of nitrogen for approximately 30 min to degas the solution. Once the reaction mixture reached 70°C, polymerization was initiated through addition of a filtered and degassed 100 mL aliquot of 0.912% w/v ammonium persulfate. The reactor was again sealed under a nitrogen blanket and allowed to react for 6 h at 70°C. Upon completion, the reaction mixture was allowed to cool to room temperature overnight with slow mixing. After cooling, the reaction solution was extracted via the bottom release valve of the reaction vessel and filtered through glass wool. The filtrate was collected into four 1 L glass bottles and stored at ~ 8°C until further purification.

Ultra-low cross-linked microgel purification

To remove linear polymer contaminants, the microgel suspension was purified using a hollow fiber TFF system consisting of the Minimate™ EVO TFF system equipped with a 0.2 µm MidGee™ Hoop microfiltration TFF cartridge. This cartridge utilizes hollow fiber filter membranes (Fig 1A), which allow small molecules to pass through while retaining the microgels. A constant volume diafiltration process (Fig 1B) was employed, in which water was added to the microgel suspension at the same rate as the permeate containing soluble linear polymers, unreacted monomers and other impurities, which were subsequently expelled into waste. The filtration process was optimized by adjusting the back pressure and pump rate to achieve a high throughput while maintaining particle purity; typically, a pump speed of 75 mL/min was used, which produced an average inlet pressure of 0.7 bar (10 psi / 0.07 MPa). The outlet pressure was adjusted until the outlet pressure gauge needle moved slightly off the zero pin. It is optimal to maintain a high pump speed without exceeding 0.7 bar (10 psi / 0.07 MPa), as the soft microgels are more likely to clog the filter at lower speeds. This process required approximately five sample volume equivalents of water to achieve the desired purity, which at typical flow rates was complete in ~ 1.5 days. Compared to equilibrium dialysis and centrifugation, TFF significantly reduced processing time, and also achieved high purity levels and improved handling convenience.

Fig 1. Use of hollow fiber filtration for removal of contaminants. (A) Hollow fiber filtration allows linear polymer contaminants to be expelled to permeate. (B) Constant volume diafiltration process.

Analysis

During the constant volume diafiltration process, the microgel suspension was periodically assessed for purity by placing a drop of the filtrate onto a glass microscope slide, drying it on a hot plate, and inspecting for polymer residue. Particle purity was further verified using an atomic force microscope (AFM) for microgel suspensions pre- and post-purification. To achieve this, a drop of the microgel solution was placed on a glass coverslip and allowed to adsorb for 5 min before gently rinsing with water and drying with air. The sample was then analyzed using the AFM.

Fig 2. Amplitude retrace AFM images of 90:10 NIPAm/AAc particles before (A) and after (B) tangential flow filtration.

Results and discussion

The amplitude retrace AFM image of the microgels before TFF purification (Fig 2A) shows microgels ~ 1 µm in size, with significant residual linear polymer contamination, which results in poor definition/fidelity in the microscopy image. The purification process via TFF was highly effective in removing these byproducts, as seen in Figure 2B, where the microgels exhibit well-defined borders and a more uniform particle size. After filtration, the microgel particles retained their characteristic appearance with minimal disruption to their size and shape, indicating that TFF does not damage the particles. The absence of linear polymer contaminants was further confirmed by placing a drop of the filtrate on a microscope coverslip. Only a faint halo of residue remained, indicating that the TFF process successfully purified the microgels. We have found in separate studies that this rapid evaluation of purity is equivalent to traditional methods such as measuring the filtrate conductivity.

Conclusions

Hollow fiber proved to be a highly effective method for purifying ULC microgels, successfully removing linear polymer contaminants while preserving particle integrity. Compared to traditional methods, hollow fiber significantly improved processing time, scalability, and purity. ULC microgels purified using hollow fiber TFF are well-suited for high-precision applications in biomedical applications such as targeted drug delivery and tissue engineering.

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