Once the full qualifying amount has been invested or irrevocably committed, which means after the last closing needed to reach the amount. Filing on the strength of a lot still under contract is premature.
The last closing sets the filing date
Hypothetical example: a Frontenac Islands landowner's first two lots close in spring and the third is under contract for September, and the regional center is pressing him to subscribe before a project deadline. The investment must be fully made, or actively in the process of being made with an irrevocable commitment, when Form I-526E is filed; money he does not yet have from a September closing cannot be counted, and a subscription agreement he cannot fund is not an investment. The safer sequence is to complete the third sale, consolidate the proceeds, pay the tax reserve into a separate account, wire the capital and fee, and file with the wire confirmation in hand.
If the project's deadline cannot wait, a bridge loan secured on assets he still owns can supply the capital, with the loan documented as personal indebtedness; a loan secured on the investment itself would not count. Processing times for Form I-526E are long, so a few months' delay at filing rarely changes the outcome.