Downstream processing, and the chapter in order

Not yet a medicine

A bioreactor full of culture is a thick mix of cells, medium, wastes and a little product. You cannot give that to a patient. The product must be taken out and cleaned. NCERT says that after the biosynthetic stage, the product has to go through a series of processes before it is ready for sale as a finished product.

Downstream processing

These processes together are called downstream processing. The word downstream means after the culture, as a stream of water goes on after a dam. The steps are as follows.

1. Separation. The product is separated from the cells and the medium.
2. Purification. The product is cleaned of the other molecules.
3. Formulation. The product is mixed with suitable preservatives, so that it lasts, and it is made into a form that can be used.
4. Quality control and clinical trials. The product is put through strict quality control tests. A drug is also put through clinical trials, which are tests on people, before it is sold.

The processing is different for every product. The steps depend on what the product is and where it is used.

Yield: what is left

Each step loses a part of the product. If the steps keep the shares $y_1$, $y_2$ and $y_3$, the overall share is the product of the three, not the mean. For example, 90 per cent, 80 per cent and 95 per cent keep $0.9 \times 0.8 \times 0.95 = 0.684$, which is 68.4 per cent. Starting with 500 g, the finished product is 342 g.

You should see that the overall share is lower than the smallest share of any one step.

The whole chapter in order

Here are the steps of recombinant DNA technology, from the DNA to the product.

The usual traps

• DNA moves to the anode. The smaller fragment moves farther.
• Ligase joins. Restriction enzymes cut. Exonucleases work from the ends.
• A palindrome reads the same on the two strands, both read from 5 prime to 3 prime.
• A vector has few cloning sites, best of all one.
• A marker finds the transformants. Insertional inactivation finds the recombinants.
• In blue and white screening, white colonies are the recombinants.
• Taq polymerase survives heat. It comes from Thermus aquaticus.
• A sparged bioreactor has sterile air bubbled through. A stirred-tank has a stirrer.
• The order of PCR: denaturation, annealing, extension.

Try it in Python

The program follows the product through the three steps of downstream processing, and gives the overall yield.

Practise with new numbers

Common mistakes

• Taking the mean of the yields. The steps follow one another, so the shares multiply.
• Thinking that the product is finished in the bioreactor. It is only made there. It must still be separated, purified, formulated and tested.
• Leaving out the clinical trials. A drug is also tested on people before it is sold.
• Mixing up the order of the steps. Select the recombinants before the culture is grown in large amounts.

Remember these

• Downstream processing: separation, purification, formulation with preservatives, quality control and, for a drug, clinical trials.
• The overall yield is the product of the yields of the steps.
• The order: isolate, cut, separate, amplify, ligate, transform, select, grow, process.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. What is downstream processing?

2. Why are preservatives added?

3. What is the extra test that a drug must pass?

4. Three steps keep 80 per cent, 90 per cent and 50 per cent. What per cent is left at the end?

5. In which order do the steps of selection and of growth in a bioreactor come?

References

• NCERT (2023). Biology, Textbook for Class XII, chapter 9, Biotechnology: Principles and Processes.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.