Avoiding a PCR Reagent Crunch: The Role of Preparative HPLC Columns

Nick Zampa

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Reading Time: 6 minutes
PCR Primer Probe

PCR Primer ProbeCritical SARS-CoV-2 PCR based diagnostics have not been immune to the supply chain disruptions so common during the COVID-19 pandemic. While the biggest disruption for PCR based testing was RNA extraction kits, enzymes and PCR primers have been affected as well. At Waters, we’ve kept a close eye on the role that high-quality preparative HPLC columns play in the PCR primer and probe supply chain. As we forge ahead through this pandemic and beyond, we will continue to help ensure adequate quality and supply of PCR primers and probes for diagnostics and routine use.

Background on RT-PCR primers and probes

Timely diagnosis of SARS-CoV-2 viral infection has been paramount to managing the novel coronavirus pandemic. In fact, COVID-19 taught us that early availability of quality diagnostics is critical to successful pandemic management in general.

Many COVID-19 assays with emergency use authorization in the U.S. and E.U. employ RT-PCR diagnostic kits, which use oligonucleotides as primers and probes during amplification and detection.

  • Fluorescent RT-PCR Probes (dual labeled): RT-PCR probes are oligonucleotides that bind to a specific target sequence of interest. A reporter fluorophore is attached to the 5’ end of the probe, while a quencher is attached to the 3’ end. The proximity of the quencher and fluorophore inhibits fluorescent signal. During amplification, a reverse transcriptase transcribes DNA from the 5’ to 3’ end. This releases the fluorophore from the probe and quencher, resulting in a measurable fluorescent signal.
  • PCR primers: PCR primers are short single-stranded oligonucleotides that bind ahead of the target sequence of interest. DNA polymerases, including reverse transcriptase, can only transcribe DNA from an existing strand in the 5’ to 3’ direction, and not create new DNA from a template strand alone. Thus, oligonucleotide primers are required for a PCR reaction.

Preparative HPLC columns and RT-PCR diagnostics

Understanding the importance of preparative HPLC columns for manufacturing RT-PCR diagnostics can be divided into two questions.

What is the most important performance parameter for an RT-PCR diagnostic test during a pandemic?

It’s sensitivity!

Avoiding false negatives is critical during a pandemic. Controlling disease spread requires that we do not erroneously diagnose patients as not having the disease, when in fact they do. As a result, the most important performance parameter for a diagnostic test is sensitivity. Analytical sensitivity and sufficiently low limits of detection (LoD) are required to ensure minimal false negatives.

PCR Primer Probe 2

So, what do preparative HPLC columns have to do with sensitivity?

It’s about purity!

As I’ve learned while speaking with our customer collaborators, reversed phase HPLC purification (primarily with ion-pairing) is recommended to achieve the primer and probe purity necessary for sufficiently sensitive and accurate PCR based diagnostic tests. Too many N-1 failure sequences result in a reduction in sensitivity. The wrong base in your primer and it is not specific to the target of interest. Free fluorescent label and your background noise increases. Missing fluorophore or quencher from a percentage of probes and sensitivity might be reduced.

While requirements for purity vary from test-to-test and company-to-company, purity >85% is often requested. Through a quick search of some of the world’s leading oligonucleotide manufacturers, you will find expert guidance on the purification techniques for different types of PCR primers and probes. Almost universally, HPLC purification is recommended for sensitive PCR assays, especially those used as diagnostics.

Ultimately, diagnostic kit manufacturers seek to reduce risk of PCR amplification failure due to poor quality or impure primers and probes. While diagnostic performance is a combination of parameters beyond purity, purity is one factor that can be controlled in the design of test kit.

Waters preparative HPLC columns and PCR primer and probe supply

We can address the link between PCR primer and probe supply and Waters preparative HPLC columns by answering three questions.

What is the link between preparative HPLC columns and primer/probe supply chains?

By enabling manufacturers to meet the high purity requirements for diagnostic PCR probes, preparative RP-HPLC columns are a critical component of ensuring adequate supply of sensitive PCR based diagnostics for COVID-19. Because of this, use of high-quality columns, capable of achieving the needed purity and providing sufficient yield is desirable. HPLC is often chosen over cartridge-based formats due to the higher capacity and better resolution (resulting in better purity) associated with columns. This combination of higher capacity and purity may be essential for urgently churning out high-demand material in a pandemic.

OK, so what could go wrong with purification that could impact my supply?

  • Imagine a column voiding unpredictably or failing mid-run. You lose your run. You lose your samples. You’re delayed.
  • Imagine you’ve developed your method at analytical scale, applied good scaling rules, and suddenly, performance at preparative scale is a nightmare. Back to the drawing board. More delay.
  • Imagine receiving a new column from the same or different lot as a previous column. You start your run and notice that it doesn’t perform the same. You might need to re-develop or optimize your method to account for this increased variability. Delayed again.
  • Imagine ordering a column only to hear of supply delays from your vendor. Will you ever get this material out the door?

Delays in production could result in delays in patient testing. In times of urgent need, you don’t have time to deal with these challenges. You want to get it right the first time, every time.

Great, but what can Waters offer to help me meet my deadlines?

At Waters, we strive to reduce your exposure to the risk of these failures by providing you with high-quality, innovative, and available solutions. Providing assurance against each challenge above requires a dedicated solution.

  • Premature voiding and short lifetimes: A packed bed must be sufficiently dense to withstand the compressive fluid forces encountered during use. As column diameters increase, it is increasingly difficult to reach the bed density required for stable, long-term performance. By employing patented OBD™ technology and packing processes, we can pack columns for each particle type and geometry with predictable and uniform densities across the length of the column. No unpredictable and premature voiding or failure.
  • Poor scaling: Even when preparative columns use the same particle and chemistry as analytical columns, good scalability is not necessarily assured due to differences in bed density. Waters OBD preparative columns are designed such that the prep scale bed densities match the analytical scale bed densities. With a range of scalable particles, we can help ensure a method scales correctly the first time, every time.
  • Irreproducibility and supply: Waters owns the entire supply and manufacturing chain after raw materials for our main columns. This affords robust process quality control across the entire column manufacturing process.
    • We produce frequent small batches of media from raw materials
    • We design, machine, and assemble our own columns
    • We pack our own columns

This also reduces our risk of unforeseen supply chain disruptions. And if those disruptions were to occur, it enables us to rapidly adjust stock. As your partner, when we burn down our supply and quality risks, so do you. Can we guarantee stock or rapid turnaround of any column, at any time? No, of course not. But are we set up to mitigate supply chain risks for you? I think so.

We strive to provide you with consistently available product of the highest quality and performance when the need is most urgent.

Register now for our COVID-19 Spotlight Series Webinar (10 a.m. ET, March 31, 2021) – Martin Gilar will explore ion pair reversed-phase chromatography for oligonucleotides including purification for PCR probes

 

 

Additional information and resources:

Have an idea, question, or collaborative proposal? We want to hear from you! If you don’t yet know your local Waters contacts, connect with the COVID Innovation Response Team for a scientific inquiry or to propose a collaboration.