The iFormulate protocol is easy to follow as illustrated below. Steps include ADD, STRESS, COLLECT DATA and Email data to data@iformulate.net for the free DOE analysis and report.
1.
Reconstitute the lyophilized formulations with 160 uL of MilliQ or WFI. To speed up reconstitution, place the plate at 40-50 C oven for about 10-15 min. Seal to prevent evaporation.
2.
ADD your protein (preferably in a simple solution with minimal formulation excipients so that the iFormulate formulations do not get influenced, PBS is OK as long as it is diluted into the formulations.
3.
Typically, 40 ul of a PBS formulation is added to each of the 25 wells from a stock concentration of around > 5 mg/ml (dilution of protein and its formulation is 5 X). This gives you 1 mg/ml protein solution in each well.
4.
Lower concentrations can be used depending on your analytical method. Total volume per well must be 200 uL.
5.
STRESS the protein in various formulations (common stress is heating 5-10 C below the melting temperature until it is about 30-50 % destabilized). DO NOT OVER STRESS as that is meaningless.
6.
COLLECT data from each well using your stability-indicating assay (e.g. protein aggregation, bioactivity, integrity, content, conformational, Tm’s, etc.)
7.
EMAIL data to htd[@]htdcorp.com and RECEIVE a DOE analysis report on Pareto analysis (ranking of key variables), optimal pH, ionic strength, stabilizer concentration, and buffer concentration.
8.
Each plate comes with ONE DOE analysis report (powerpoint). For additional DOE analysis, there is a charge of $ 500.00
9.
You can then validate the lead formulations proposed from the DOE data. THIS IS VERY IMPORTANT. It validates the Design Space based on your empirical data from iFormulate.
10.
You can use the extra rows on G2-6, or G7-11 to have formulation controls: e.g. 40 uL protein diluted in 160 uL of WFI or current protein formulation.
In action
Please see the posters and technical papers on how iFormulate is used in antibody formulation development.
The concept of iFormulate was developed at HTD Biosystems. In formulating many proteins, an efficient high throughput algorithm was designed to perform preformulation of proteins in a rational fashion using Design of Experiments (DOE) and multivariate analysis.
