scott.pritchett

sgRNA is arguably the most critical raw material used in the development of in vivo CRISPR-based gene editing therapies. The amount of sgRNA used is directly related to editing efficiency; more sgRNA leads to higher efficiency. The purity of sgRNA also affects its required amount. Higher purity means less sgRNA is needed, reducing costs. Additionally, fewer impurities decrease the risk of side effects from unknown substances. Therefore, improving the purity of sgRNA and tracking any remaining impurities are critical to the development of FDA-regulated products that utilize sgRNA.

FDA’s recommendations regarding the purity and impurity of sgRNA are as follows:

- Purity of gRNA full length product should be ≥80%, and
- identify any impurities that are present ≥1%.

Note: FDA acknowledges that purity acceptance criteria can be dependent on the sensitivity of the analytical method used.

Scaling up with upgraded purification methods

Some in vivo CRISPR gene editing therapies will involve gram-level production of sgRNA. For example, the FDA approved two in vivo CRISPR gene editing therapies in 2023, NTLA-2001 (NCT05697861) and NTLA-2002 (NCT05120830), developed by Intellia Therapeutics. NTLA-2001 employs lipid nanoparticles containing mRNA encoding Cas9 and single guide RNA (sgRNA) targeting the liver-specific TTR gene. NTLA-2002 delivers the CRISPR-Cas9 gene editing system in the form of mRNA using LNPs, targeting the KLKB1 gene to permanently reduce plasma kallikrein enzyme activity, thereby preventing hereditary angioedema (HAE) attacks. Based on these application scenarios, in 2022, a gram-level sgRNA production platform based on the AKTA oligopilot plus 100 (OP100) system was established. In 2023, cGMP production of gram-level sgRNA was achieved.

When the synthesis scale is expanded from nmol level(ug) to mmol level (multigram quantities， OP100 platform), the types and volume of impurities will both increase. sgRNA impurities can be roughly divided into the following two categories: early-elute impurities and late-elute impurities. Early-elute impurities include N-X, desulfurization (PO), depurination, depyrimidination. Late-elute impurities, which can be notoriously difficult to remove, include N+X, partial-protected impurities, non-resolvable impurities. Removing these impurities requires optimizing the synthesis process and purification process. Different chromatography methods can be leveraged for the purification of sgRNA.

Commonly used RNA purification methods

Anion exchange chromatography (AEX) and ion pair reverse phase liquid chromatography (IP-RP) are two of the most used purification methods for RNA isolation.

Anion exchange chromatography (AEX):

AEX is a technique that uses the adsorption capacity of ion exchange resins for negatively charged molecules to separate and purify biomolecules. In RNA purification applications, its principle is based on the interaction between the negative charge of the phosphate group on the RNA molecule and the positively charged group on the anion exchange resin. By gradually increasing the salt concentration in the eluent to change the ionic strength, the electrostatic force between the RNA and the resin is weakened, causing the RNA to dissociate from the resin and flow out with the eluent. Since different RNA molecules may have different lengths and secondary structures, their affinity to the anion exchange resin may be different. Thus, different RNA molecules can be graded and eluted by simply adjusting the salt concentration.

Ion pair reverse phase (IP-RP) liquid chromatography:

IP-RP is a technique used to separate hydrophilic or charged analytes on columns using reversed-phase or “neutral” stationary phases that do not carry charges. IP-RP high performance liquid chromatography (IP-RP-HPLC) involves modifying the polarity of charged analytes by interacting with an ion-pairing reagent (such as TEA, DEA, TPA, HA, DBA) added to the mobile phase.

The principles of IP-RP-HPLC for RNA purification include:

(1) Using the hydrophobic stationary phase of reversed-phase chromatography.
(2) Adding ion-pairing agents to enhance the hydrophobicity of RNA and improve its retention on the stationary phase.
(3) Optimizing RNA elution and separation by adjusting the composition of the mobile phase.

Hydrophobic chromatography (HIC):

HIC is an additional method that can be used to perform DMT-on purification for RNA.

HIC separates molecules based on their hydrophobicity. Dimethoxytrityl (DMT), a 5’protecting group, is used in RNA synthesis to temporarily mask the characteristic chemistry of a 5’-hydroxy functional group. DMT can be left on the 5’ end of RNA after synthesis. This enables purification by HIC since the DMT-on group is strongly hydrophobic. In our process development, HIC is used to remove short RNA without DMT, while RNA with DMT is retained on the HIC column. After removing DMT, IP-RP-HPLC is used to produce pure RNA with high purity.

At GenScript, we use both HIC and IP-RP-HPLC to achieve high purity sgRNA.

Improving sgRNA purity to serve our customers better

In order to understand the characteristics of our sgRNA products, we compared the quality of our cGMP sgRNA with that of other well-known cGMP nucleic acid manufacturers (Figure 1). The purity was 84.03%, the early-elute impurities were 5.74% and late-elute impurities were 10.23%. Before process optimization, our sgRNA was not much different from other mainstream GMP suppliers.

To provide our GMP customers with the highest purity product possible, we have continuously optimized our purification processes. One of our most significant optimizations includes a unique sgRNA purification process that we designed on the OP100 platform. After the crude product is subjected to HIC purification and TFF, it is then subjected to IP-RP-HPLC purification. When performing IP-RP-HPLC purification, the most used ion pair reagent is TEAA. After the sample is purified by TEAA, most of the early-elute impurities and late-elute impurities can be removed, but only a very small amount of them cannot be removed. While continuing to optimize the pH, gradient, and time of the mobile phase will not have a big effect on improving purity, we found that optimizing the composition of the ion pair reagent can greatly reduce a certain type of impurities.

We developed three ion-pair reagents for purification, which we will refer to as GS-P1, GS-P2 and GS-P3. Compared to GS-P1, GS-P2 significantly reduced non-resolvable impurities and part of n+1 impurities. Compared to GS-P2, GS-P3 improved the recovery rate. Therefore, we compared the purification efficiency of GS-P1 and GS-P3. GS-P1 and GS-P3 showed different separation capabilities for late-elute impurities. GS-P3 has better impurity separation ability, reducing the impurities from 23.10% to 4.34%, and increasing the final purity from 75.07% to 90.86% (Figure 2).

Purification tests of different sequences (S2 and S3) were also performed using GS-P1 and GS-P3, and the results are shown in Figure 3. S2 is a sample with higher synthetic quality, while S3 is a sample with more modifications. For S2, not only did the recovery rate improve from 19.87% to 31.07%, but the late-elute impurities were also reduced from 11.21% to 1.11% (Figure 3), indicating that GS-P3 has better purification performance. For S3, not only did the recovery rate improve from 15.70% to 37.26%, but the late-elute impurities were also reduced from 13.72% to 6.25% (Figure 3), indicating that GS-P3 has better purification performance.

Mutation analysis using Next-generation sequencing

Traditional methods like UPLC and MS/MS have limitations in detecting sequence variants, particularly in long-guide RNA. Next-generation sequencing (NGS) can be employed for the analysis of mutation profiling of sgRNA, due to the high throughput and precision offered by the method. When we analyzed mutations in the S2 and S3 sequences at every base by NGS, the results showed a very low mutation ratio of <1% (Figure 4), and thus confirmed the high quality of our sgRNA.

As a result, we selected the GS-P3 ion-pair reagent for use in our purification method for producing gram-level sgRNA under the cGMP system. GS-P3 has the following characteristics:

(1) Regardless of crude product quality, it has a strong ability to remove early-elute impurities and late-elute impurities.
(2) It has a weak correlation with the sequence and a variety of different sequences can achieve high purification recovery rates and purities.
(3) It does not require heating and can be purified at room temperature.

By leveraging advanced purification techniques and continuous optimization, GenScript is committed to delivering the highest quality sgRNA, helping to ensure the success of our customers' CRISPR gene editing projects at every stage of their research.

Learn more about our GenCRISPR Synthetic sgRNA services here.

We can also meet customers' GMP requirements for gram-level sgRNA. Our GMP production includes the following capabilities:

• Upgraded synthesis and purification process ensures purity ≥80%.
• Multi-dimensional quality characterization methods: results in sgRNA quality that is superior to that of other GMP vendors.
• Reliable quality management system: supply of sgRNA is compliant with cGMP guidelines, which is suited for preclinical, IND enabling and Clinical Trials Phase I/II use.
• Customizable services: supports a variety of delivery formats (liquid/lyophilized powder).
• Capacity and TAT: Our platform yield is 1.32-1.82g/mmol for 100nt sgRNA and our production time is ～25days/batch.

Learn more about our GenCRISPR cGMP sgRNA services here.

References:

1. Jonathan D. Finn, et.al. A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing, Cell Reports 22, 2227–2235, 2018

2. Anastassia Kanavarioti, HPLC methods for purity evaluation of man-made single-stranded RNAs, Scientific RePorts, 2019, 9,1019, DOI:10.1038/s41598-018-37642-z.

3. Torgny Fornstedt, Martin Enmark, Separation of therapeutic oligonucleotides using ion-pair reversed-phase chromatography based on fundamental separation science, Journal of Chromatography Open 3 (2023) 100079.

4. Jose V. Bonilla, G. Susan Srivatsa, Handbook of Analysis of Oligonucleotides and Relate-eluted Products, 2011.

5. Yesi Shi, et. al. Chemically Modified Platforms for Better RNA Therapeutics Chem. Rev. 2024, 124, 929-1033.

6. Gavin, D., Kwilas, A., Sanduja, S., & Imperato, G. (2024, February). FDA CBER Webinar: Human Gene Therapy Products Incorporating Human Genome Editing. Lecture, Virtual Webinar; Virtual Webinar.