Sterility testing.

Question 1

What is sterility?

Answer 1

It is defined as the freedom from the presence of viable microorganisms. Conditions for true sterility are way too harsh for active ingredients so it must be defined in functional terms. Sterility ties into many facets of good manufacturing practises.

Question 2

How are containers tested for sterility?

Answer 2

A container is sterile when there is less than a 1 in 1 million chance that it is contaminated with a replicating organism. Containers are tested periodically rather than every container during filling operations.

Question 3

How do we test for specific contaminants?

Answer 3

We can do nucleic acid amplification where either Polymerase Chain Reaction, Transcription Mediated Amplification or Loop-mediated Isothermal Amplification.

Question 4

How does PCR work?

Answer 4

It goes through cycles. Sample DNA is heated to 90 degrees to denature it and separate the strands. It is then cooled to 55-65 degrees to anneal the primers to the target sequences. It is then heated to 72 degrees to allow for taq polymerase to bind and extend the sequence from the primers. This is then repeated until enough amplicons are yielded. The reaction must contain free DNA nucleotides to allow for this extension.

Question 5

How does TMA work?

Answer 5

It is a reaction that occurs at a constant temperature of 40-55 degrees. Essentially, regardless of the sample input, primers with a functional T7 promoter must be annealed and a reverse transcriptase will generate first strand of cDNA or RNA DNA hybrid whilst the original template is degraded by RNase H activity of the reverse transcriptase. The reverse transcriptase then produces a second strand to generated dsdna with a functional T7 promoter. Then the T7 RNA polymerase can bind and generated multiple copies of RNA.

Question 6

How does LAMP work?

Answer 6

It is a reaction that operates at a constant temperature of 60-65 degrees. It uses a strand displacing DNA polymerase and multiple distinct primers, typically two inner and two outer. It begins with the generation of a dumbbell structure which act as starting points for rapid, auto-cycling amplification producing cauliflower like DNA concatemers. The polymerase displaces the newly synthesised DNA to leave room for further amplification.