Twumasi netted with two minutes left on the clock and after the Spanish had taken a comfortable 3-1 lead thanks to a brace from Congolese attacker Cedric Bakambu.
Astana went into the game a win away from sealing qualification to the next stage and they made a bright start.
Congo forward Junior Kabananga put Astana in front on 22 minutes but Villarreal equalised before halftime.
Bakambu added second and third for Villarreal before Twumasi pulled a goal back for the Kazakhstan champions.
The result means Astana have to win their last match to qualify.
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