Marin Anicic’s 38th minute strike broke the hearts of the home side (Slavia Praha) at the Eden Aréna in Praha.
Slavia Praha needed just a draw to qualify to the next round of the competition but missed out after the painful defeat to the Kazakh Champions.
Twumasi, who scored four goals in the Group stage was at his usual best for Astana as he played almost the entire duration before making way for Djordje Despotovic in additional time.
Astana finished second behind Villarreal who topped Group A with 11 points.
The Ghanaian international and his side now awaits the draw on Monday to know their next opponent in the round of 32.
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