Presented by Keith Jarvis, Senior Director, Process Chemistry, Editas Medicine
Keith Jarvis from Editas Medicine began his talk on CRISPR Based Cell Therapy production with a description of how ex vivo therapies work. Ex vivo therapies begin with the removal of cells from an individual patient, editing them and re-introducing them. In cell therapy, a universal cell population may be edited and then can be provided to any patient without the need to donate cells. He explained that two programmable proteins for editing cells (target and edit specific genes in DNA) are Cas12a (previously called cpf1) and Cas9. These two nucleases each pair with guide RNA (gRNA) that in turn recognises a specific DNA site where it cuts a double stranded break. The gRNA for Cas12a has distinct motifs adjacent to the protospacer and cuts the DNA in staggered manner resulting in greater gene repair efficiency and accuracy.
Synthetic forms of the gRNA (AsCas12a) have lowered risk of off-target editing and have proven more specific than SpCas9 across matched sites in the genome. In gRNA, purity and yield drop with increasing length. Generally, Cas12a associated guides are preferred as they are much shorter than Cas9 associated guides (40-60-mer versus ~100mer), and a lack of sequence fidelity may lead to unanticipated off-target editing. Synthesis occurs in 3’-5” direction, and Cas9 is sensitive to mismatches at the 5’ end where sequence fidelity is lower, whereas Cas12a is most sensitive to mismatches at the 3’ end where sequence fidelity is higher as this is the start of the synthesis.
The active pharmaceutical ingredient (API) is the ribonuclease protein complex (RNP), a combination of nuclease Cas12a and its gRNA.. The patient-engineered cell may be considered the active product. Both gRNA and Cas12a are defined as critical starting materials. Once synthesized, the gRNAs contain regions of RNA and DNA, 2’O-methyl modifications, phosphothioates, phosphodiester backbones and are 40-60 nucleotides in length. During synthesis of longer strands, a small loss in coupling efficiency translates to large drops in yield (e.g., a 99% efficiency produces a 36% drop in yield for a 100 mer).
Jarvis outlined the production plan at Editas, which includes in-process testing points for the crude-workup, purification, ultrapurification, suspension and batch pooling, and the sterile filtration stages. Their in-house process development laboratory will be used to synthesize the crude product, which will then be sent to the clean room for the downstream processing step from purification through final fill and finish. All in process analysis will be performed by Editas while final release testing will be performed at an external contract facility. He shared that standard analytical methods will be used for the gRNA including identity with mass spectrometry, strength with UV spectrometry, and purity by U-HPLC. Impurities will be identified as early and late eluting clusters. Standard testing will be performed at a good manufacturing practices (GMP) contract research organization (CRO) including safety, general, identity, strength, purity, and impurities.
Jarvis explained that quality systems for GMP include a materials management system, production records, cleaning program, gowning qualification a program, environmental monitoring program, Installation Qualification /Operational Qualification (IQ/OQ) for all equipment, data backup, computer system validation for GMP equipment and networks, quality control for in process testing in-house and release testing at a contract facility. Internal GMP manufacturing will accelerate the preclinical programs, with the advantage that it can optimize at the appropriate scale and will not require 6-12 month wait for a CMO slot. It will allow for immediate reaction to evolving research programs, and all IP will be retained internally.
Keith Jarvis is the Senior Director of Process Chemistry at Editas Medicine
