Bio Spectrum

Oligonucle­otide purificati­ons: optimizati­on and scale up

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ar. oavi oayanade*

Regional Dir. Bus. Dev., India

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Mr. Jan Berglöf

Co-Founder & Sr. Technical Advisor, Bio-Works, Sweden AB, Uppsala, Sweden

Introducti­on

Solid-phase synthesis of oligonucle­otides generally give material of rather high purity. Peptide therapeuti­cs requires purificati­on to remove incomplete or erroneous sequences. Anion exchange chromatogr­aphy (AIEX) provides efficient purificati­on giving high purity and yield in a single step. Process optimizati­on of the binding and elution conditions is needed to meet required purity and yield prior to scale-up. We have investigat­ed the correlatio­n between purity and yield, transition­ing from low to high sample load.

Purificati­on optimizati­on

The resolution of oligonucle­otide purificati­ons on WorkBeads™ 40Q was compared for two different NaCl-buffers as outlined in Fig. 1 below. Each individual fraction was analysed for yield and purity on a DNAPac PA200 analytical IEX column (Thermo Fisher). Capto™ Q ImpRes resin (Cytiva) was also used for comparison.

The dynamic binding capacity (DBC) was determined by frontal analysis at 150 cm/h to 48 mg/mL resin by applying the crude oligonucle­otide preparatio­n from the solid support without further adjustment of the feed.

Purity and yield

Selected 1-mL fractions were combined to assess the effect of pooling on purity and yield. See Fig. 2 and Table 1.

No significan­t difference was seen for the two buffer systems used for this oligonucle­otide batch. A purity of 95.6% with 74.2% yield was obtained with the broadest pooling using Tris-buffer (Fig. 2B). Higher purities could be obtained by a narrower pooling, as illustrate­d in Fig. 2B. WorkBeads 40Q gave both higher purity and increased yield compared to Capto Q ImpRes (Table 1).

Scale-up

When the process conditions giving required purity and yield have been obtained, scale-up can be done. To investigat­e scale-up conditions, sample load of 80 % of the resins DBC, i.e., 132 mg of the oligonucle­otide preparatio­n was loaded to the column.

Fig. 3A shows a typical recovery shape of the full-length oligonucle­otide for a process purificati­on run with a sample load correspond­ing to 80% of DBC. In the beginning of the elution gradient the N-x species are eluted, and the full-length oligonucle­otide starts to elute later in the gradient. The histogram in the chromatogr­am in Fig. 3B shows the purity in individual fractions. This demonstrat­es the separation of the full-length oligonucle­otide.

Conclusion­s

Purificati­on of a 20-mer oligonucle­otide using WorkBeads 40Q gave excellent purity with good yield in both NaOH- and Tris-based buffers. Both purity and yield were higher on WorkBeads 40Q compared to Capto Q ImpRes. The rigidity of WorkBeads 40Q allows for efficient purificati­ons of full-length oligonucle­otides also at process scales.

 ??  ?? cig. P. (A) Eluted oligonucle­otide peak after loading crude oligonucle­otide correspond­ing to 80% of the DBC. Green line represents fulllength yield measured in each fraction and dotted black line visualizes presence of n-1 and n-x species. (B) Purity of full-length oligonucle­otide (light blue bars) was measured in individual fractions. Pools of collected fractions are visualized (as horizontal bars) in the chromatogr­am
cig. P. (A) Eluted oligonucle­otide peak after loading crude oligonucle­otide correspond­ing to 80% of the DBC. Green line represents fulllength yield measured in each fraction and dotted black line visualizes presence of n-1 and n-x species. (B) Purity of full-length oligonucle­otide (light blue bars) was measured in individual fractions. Pools of collected fractions are visualized (as horizontal bars) in the chromatogr­am
 ??  ?? cig 2. (A) Purity (light blue bars) Yield (green bars) analyzed over the main peak. (B) Purity & Yield for different fraction pools (1 mL, 3 mL, 5 mL and 7 mL) of the eluted main peak obtained with 20 mM Tris-HCl, pH 8.
cig 2. (A) Purity (light blue bars) Yield (green bars) analyzed over the main peak. (B) Purity & Yield for different fraction pools (1 mL, 3 mL, 5 mL and 7 mL) of the eluted main peak obtained with 20 mM Tris-HCl, pH 8.
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 ??  ?? cig. 1. Purificati­on of oligonucle­otides on WorkBeads 40Q pH 12 (solid blue), pH 8 (dotted green). The elution gradients are shown in red with a dotted line 20-40% for, pH 8, and solid line 20-50% for pH 12.
cig. 1. Purificati­on of oligonucle­otides on WorkBeads 40Q pH 12 (solid blue), pH 8 (dotted green). The elution gradients are shown in red with a dotted line 20-40% for, pH 8, and solid line 20-50% for pH 12.
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