Table 1.

Proposed qualitative framework for the assessment of the criticality of process parameters (PPs) based on the qualitative assigned impact of PPs on the CQAs^a.

Process parameter	RNA sequence integrity	RNA sequence identity	RNA yield	Sourceb	Classification
Temperature	−2	−1	±3	Data, Expert51	CPP
Pressure	±1	±1	±1	Expert	PP
Mixing	±1	±1	±1	Expert	PP
Reactor dimensions	0	0	0	Expert	PP
Reaction time	±2	0	3	Data, Expert	CPP
pH in transcription reactor	±2	±2	±3	Data, Expert52,53	CPP
DNA template sequence	−2	−3	0	Expert	CPP
DNA template concentration	0	0	+2	Data, Expert	CPP
T7RNAP concentration	±1	±1	+3	Data, Expert	CPP
5’ cap analogue concentration	0	±3	0	Data, Expert	CPP
Total Mg concentration	0	±2	±3	Data, Expert44	CPP
DTT concentration	±2	±2	±1	Data, Expert45	CPP
Spermidine concentration	±2	±2	±3	Data, Expert46	CPP
GTP concentration	+1	±3	±3	Data, Expert	CPP
Total NTP concentration	+2	±2	±3	Data, Expert	CPP
Ratio of NTPs	+1	±3	±3	Expert	CPP

The PP-CQA relationship is characterised by an impact magnitude rating, a sign indicating directionality and shape of the CQA = f (PP) plot. The magnitude of the impact was rated from 0 (low) to 3 (high), and PPs rated with 2 or 3 were considered critical, thus critical process parameters (CPPs). The direction and type of CPP-CQA relationship were characterised either by a positive slope labelled with plus “+”, a negative slope labelled with a minus “−”, or a peak behaviour whereby the CQA increases with increasing the PP reaches a peak and then decreases, labelled with plus-minus “±”.

^aThe CQAs of “Bacterial endotoxins”, “Bioburden”, 5’ capping efficiency and “Post-filtration pH” from Supplementary Table 1 were not included in this table because these can be assumed to be well-controlled in a GMP bioproduction process.

^bThe ratings provided in the “RNA sequence integrity”, “RNA sequence identity” and “RNA Yield” columns were based on experimental data (Data), information from the literature (where applicable reference is included and are listed in the main article bibliography) and expert knowledge (Expert).