Glen Report 19.21: Glen-Pak™ Cartridges – The Ultimate Purification Cartridges

Introduction

The use of Oligonucleotide Purification Cartridges has been a popular option for purifying synthetic oligonucleotides based on the DMT-on procedure. These cartridges typically contain polymeric packing materials which are stable to dilute ammonium hydroxide or ammonium hydroxide/methylamine (AMA). The technique relies on the affinity of reverse phase packing materials for the 4,4’-dimethoxytrityl (DMT) group at the 5’ terminus of synthetic oligonucleotides. The advantages of these cartridges are clear: speed, inexpensive, no need to evaporate corrosive solutions, product elutes in a small volume of aqueous acetonitrile. However, there is also a major disadvantage of traditional cartridges in that the longest oligos that can be reliably purified by this method are approximately 50mers. In addition, the yield of purified product from traditional cartridges is normally 40-60% of theoretical and the crude oligos need to be loaded in at least two passes. This clearly makes traditional cartridges unwieldy for use in high throughput robotic work stations.

Poly-Pak™

Of the traditional oligonucleotide purification cartridges, our favorite remains Poly-Pak™ cartridges and barrels – and we are in no way biased! By using 3-5 micron polydivinylbenzene packing, we were able to produce cartridges and barrels that use the smallest wash volumes in the market. Also, the product elutes in 0.5mL of aqueous acetonitrile, making evaporation fast and simple. Poly-Pak cartridges have been a staple for oligonucleotide purification for 15 years. Unfortunately, traditional cartridges, including Poly-Paks, have the disadvantage that they require the crude oligo solution to be loaded in two passes. This is clearly unsuitable for the use of these cartridges in high throughput situations.

Fluoro-Pak™

As an example of the next generation of purification cartridges, Glen Research began in 2006 to offer a new cartridge purification system - Fluoro-Pak™ cartridges for the purification of longer oligonucleotides as a complement to the Poly-Pak range. Fluoro-Pak cartridges use the exceptionally high affinity of fluorous-tagged oligonucleotides for fluorous packing materials. In this case, the DMT group at the 5’ terminus is adorned with a fluorous ponytail, which allows simple purification of the full-length DMT containing oligonucleotide from the capped failure sequences. The Fluoro-Pak system works just fine for shorter oligos but really excels by offering excellent purification of longer oligos in the 50-100+ range. The system also offers one pass loading of the crude oligonucleotides to the cartridges, making it much more acceptable in high throughput systems. Fluorous DMT protected monomers are required for this system to work and a variety of these are available, including all four regular DNA monomers and fluorous CPR II, which may be used to purify 5’-phosphorylated oligos. However, a clear downside to this purification technique is the requirement of these special monomers, which add to the cost of purification.

Glen-Pak™

Our latest option is Glen-Pak™ cartridges, which offer the very best of Poly-Pak with the improvements offered by Fluoro-Pak rolled into one – the Ultimate Purification Cartridge. Glen-Pak cartridges:

Glen Pak
  • Are available in two formulations, one optimized for DNA purification and the other for RNA purification.
  • Extend the range of purification of Poly-Pak cartridges to 100+.
  • Use the regular DMT group as the affinity tag.
  • Are available as cartridges for syringe use.
  • Are available as cartridges for use on vacuum manifolds and high-throughput devices.
  • Require a single loading of the crude oligonucleotide solution diluted with an aqueous salt solution.
  • Can be used for desalting oligonucleotides.
  • Have been tested with amino-modifiers, biotin and biotinTEG, and phosphorylation with CPR II.
  • Have been tested with most dyes, including Cy dyes, FAM, HEX and TET.
  • Demonstrate recoveries of full-length oligonucleotide with yields typically greater than 90%.
  • Cost effective.
Table: Comparison of available cartridges
FeaturePoly-Pak™Fluoro-Pak™Glen-Pak™
Single loading - √√√ √√√
Oligo range to 100 + √√ √√√ √√√
Desalting cycle available √√√ - √√√
Variety of formats available √√ √√√
Yield of available full-length oligo √√√ √√√
Performance with modifiers √√√ √√√
Performance with dyes √√√ - √√√
Optimized for RNA √√ - √√√
Cost √√ √√√

Comparison

Salient features of all three cartridge types are collected in the Table, which shows clearly the advantages that Glen-Pak cartridges offer over traditional RP cartridges, like Poly-Pak, and even over more sophisticated systems, like Fluoro-Pak.

Examples

1. DNA Purification

Glen-Pak DNA Purification cartridges have been used with a variety of sequences, from short to long (>100mer). They are fully compatible with any DMT-on oligonucleotide, including those with a variety of labels and tags, e.g., 5'-phosphate using CPR II, 5'-amine, 5'-biotin. Although Glen-Pak cartridges are optimized for the capture of DMT groups, they are also compatible with dyes with no DMT group, e.g., Cy dyes, FAM, HEX and TET.

Figure 1 illustrates the purification capability of Glen-Pak DNA Purification cartridges with a mixed base 71mer. A small portion of the crude oligonucleotide was detritylated and run on ion-exchange HPLC to illustrate the large number of failure sequences present. The majority of the crude oligonucleotide was purified on a Glen-Pak DNA Purification cartridge and the product was also run on ion-exchange HPLC. The two chromatograms demonstrate the remarkable enhancement of purity while the recovery of full-length product was >90%.

Figure 2 shows the results of a similar experiment where a 71mer was phosphorylated with CPR II and purified on a Glen-Pak DNA Purification cartridge. Again the recovery of full-length product was >90% and the purity was substantially enhanced.

2. RNA Purification

The use of Glen-Pak RNA Purification cartridges is illustrated in the Technical Brief on Page 5. Following DMT-on RNA synthesis and regular base deprotection with your favorite basic mix, the silyl protecting groups are removed with triethylamine trihydrofluoride containing dimethylsulfoxide and triethylamine. This mixture is diluted with the RNA Quenching Buffer and applied directly to Glen-Pak RNA Purification cartridges to complete the purification step. Figure 3 demonstrates purification of an RNA 22mer using a Glen-Pak RNA Purification cartridge.

Conclusion

DMT-on purification of oligonucleotides using traditional cartridges, exemplified by Poly-Pak cartridges, has served the oligo synthesis community well for many years. However, two major deficiencies in the traditional cartridges quickly became apparent: a single loading process did not work, making them problematical for high throughput robotics systems; and the range of successful purification was restricted to around 50mers. The Fluoro-Pak purification system, as applied to oligonucleotide purification, solved both of these problems but at a higher cost. The required fluorous DMT monomers must be available to be added in the final synthesis cycle.

Glen-Pak DNA and RNA cartridges have advantages over Poly-Pak cartridges in that a single loading of the diluted crude deprotection solution is all that is necessary. Also, the range of purification has been extended to 100+ using DMT-on oligos. Glen-Pak cartridges have similar performance to Fluoro-Pak cartridges but without the need for the fluorous DMT group at the 5’ terminus, so the cost is lower. In addition, Glen-Pak cartridges allow purification of virtually the complete range of dyes and modifiers. A booklet, User Guide to Glen-Pak™ Purification describes in detail the process and several applications.

Poly-Pak™ and Glen-Pak™ are trademarks of Glen Research Corporation. Fluoro-Pak™ is a trademark of Berry & Associates, Inc.

purified hplc
Figure 2: IEX HPLC analysis of a crude and purified 71-mer 5'-phosphorylated with CPR II
Column: Dionex DNAPac PA200, 250 X 4mm Buffers: A- 10mM NaClO4, 25mM TRIS-HCl, 20% Acetonitrile, pH 7.4 ; B- 600mM NaClO4, 25mM TRIS-HCl, 20% Acetonitrile, pH 7.4 Gradient: 0-40% Buffer B at a flow rate of 1mL/min.
purified hplc
Figure 1: IEX HPLC analysis of a crude and Glen-Pak purified 71-mer
RP HPLC
RP HPLC Column: Waters Spherisorb S5 OD2 150 x 4.6 mm A: -100% ACN; Buffer B - 0.1M TEAA Gradient: 3%A to 40%A over 30 min at a flow rate of 1mL/min.
IEX HPLC
IEX HPLC Column: Dionex DNAPac PA200 250 x 4mm Buffers: A - 25mM Tris, pH 8, 5% ACN;
B - 25mM Tris, pH 8, 5% ACN, 1.0M Nh4Cl, pH 8 Gradient: 0-90% Buffer B at a flow rate of 1mL/min.
Figure 3: RP HPLC of a crude RNA 22mer and IEX HPLC analysis of a Glen-Pak purified RNA 22mer

Product Information

Glen-Pak™ Reverse Phase Purification Products