The natural molecular vaccine adjuvant QS-21 is in increasing demand for several approved vaccines, including shingles, malaria, and—outside the United States—COVID-19. More than a dozen vaccines are being developed against those conditions as well as influenza, hepatitis B virus, human papillomavirus, HIV/AIDS, tuberculosis, non-small-cell lung carcinoma (NSCLC), and melanoma. Purification, however, has been challenging.
Now, scientists at the Access to Advanced Health Institute (AAHI) say they have developed a simple and efficient method that produces high yields of QS-21 at more than 97% purity under GMP manufacturing conditions. The challenge to date has been the nature of QS-21 itself.
“Currently, the primary source of QS-21 is the bark of mature Quillaja saponaria trees, a slow-growing soap bark tree found only in a specific region in Chile. It is relatively enriched in the bark but, on an absolute scale, it’s actually in limited amounts, and those amounts vary in response to climate and environmental factors,” Yizhi (Stacey) Qi, PhD, scientist, AAHI, tells GEN.
Additionally, the natural mix of saponins is quite complex. “These mixtures have tens, if not hundreds, of other saponins that are close in chemical structure and property to the QS-21 molecule,” which is, itself, a combination of four different isomers, adds Qi. Consequently, separation is challenging.
AAHI scientists have developed a two-step orthogonal chromatographic process to improve separation, outlined in a paper published last year and since improved upon. The process involves a polar reversed-phase (RP) chromatography step followed by a hydrophilic interaction chromatography (HILIC) step.
“We’re taking full advantage of the amphiphilic—both hydrophobic and hydrophilic—nature of the saponins to maximize separation,” Qi says. “Many of the structural differences among these saponins are coming from the hydrophilic glycosyl groups, and they can be subtle. Coupling reverse phase chromatography with the orthogonal HILIC technique, well-established for carbohydrate separation, offers the all-round retention and selectivity required to simultaneously achieve high purity and yield without tradeoff. This, in turn, reduces waste of the raw material, which is in increasingly limited supply.”
Alternative purification methods typically use multiple rounds of reverse phase chromatography, which exploits the hydrophobic, non-polar part of the molecule, but may require additional steps between rounds. Some of those methods require pretreatment or have strict requirements for the composition of the starting material.
“When AAHI used its method for commercial scale GMP manufacturing runs, it produced QS-21 at the gram level,” notes Q. “A typical dose of a vaccine formulation only requires micrograms of this material,” so the gram quantities produced are enough to support a clinical trial and small- to medium-scale commercial needs.