Magazine Article | September 1, 2009

Q&A Roundtable On Recent Developments In LC Technology

Source: Life Science Leader

By Dan Schell

Discuss the recent advancements in LC (liquid chromatography) technology and the overall impact on the Pharma/biopharm market.

Helmut Schulenberg-Schell, Ph.D., worldwide marketing manager for Agilent LC Systems and Modules
The main changes have centered around power range, which is pressure multiplied by flow. Power range determines which column dimensions and particle sizes and types can be used to achieve different levels of resolution and speed. In recent years, there’s been an intense debate about the value of operating in different power range segments, with accompanying method development choices.

Over the years, different vendors developed instruments providing different pressure/flow ranges, including higher pressures to increase performance in particular areas. As of the beginning of this year, there were no fewer than 11 different instruments from 10 manufacturers in the HPLC (high performance liquid chromatography) and UHPLC (ultra high performance liquid chromatography) segment, each with a different power range.

Michael Carolus, analytical specialist, Pharmacare Limited
Nowadays most manufacturers have some sort of UPLC (ultra performance liquid chromatography)/UHPLC available, which clearly indicates the market has embraced the technology. The instruments have improved significantly, thereby allowing scientists to learn more about their products and make decisions more quickly. For example, some UV HPLC detectors have a maximum sampling rate of 10 HZ (10 points per sec) compared to UPLC detectors of more than 100 HZ.

Michael McGinley, bioseparations product manager, Phenomenex Inc.
Although some in the industry have tried to convince users that there has been some technological revolution in the HPLC world in the last few years, a global overview of the market reveals that the trend toward reducing separation time that has been occurring since the late 90s has continued in the last few years with incremental improvements in HPLC column and instrument technologies (aka UHPLC or UPLC). While decreasing particle-size materials is just a natural extension of this trend (along with high pressure systems to operate such columns), new separation media technologies, such as monoliths and solid core media, break away from this trend and offer new solutions for liquid chromatography separations using existing instrumentation.

What factors have driven advancements, and how do you see those factors changing moving forward?

McGinley (Phenomenex)
The drive by organizations to reduce costs and improve throughput has driven the need for reduced analysis times. While one can increase throughput by purchasing more HPLCs, reducing run times can deliver the same increase in throughput without any additional instrument costs. A key parameter in moving toward high throughput solutions revolves around whether or not such solutions can use existing instrumentation or if new equipment is required.

Carolus (Pharmacare Limited)
Pharmaceutical companies have been driven to bring products to the market faster by increasing sample throughput. I think this has been the biggest driving force for the advancement of LC technology over the last few years.

Dr. Rohit Khanna, vice president, worldwide marketing at Waters
Today, pharma businesses are faced with increased competition and fewer resources. Our customers consistently tell us that they can’t work harder than they currently work; they need to work smarter. They want to reduce business risk — the risk of placing the wrong bet on the wrong drug candidates, the risk of having their operations shut down due to an out-of-compliance event, the risk of losing a client by missing target delivery dates, the risk of losing out to a competitor in bringing a drug to market, and the risk of compromising intellectual property.

What do these advancements mean for the future of analytical laboratories and  analytical test development?

Schulenberg-Schell (Agilent)
Labs continuously need to be able to do more with less, while obtaining as good or better results. In managing instrument life cycles, there are emerging opportunities to standardize on a single instrument which can perform a much wider range of analytical LC methods.

Khanna (Waters)
The future impact of UPLC on analytical laboratories includes improved laboratory processes with real business benefits, elevated performance of mass spectrometry when using UPLC as an inlet, and quantifiable environmental benefits from reduced solvent use, electricity, and lab space.

Generally, how do HPLC systems differ from UPLC/UHPLC systems? Explain the major advantages and any disadvantages.

Schulenberg-Schell (Agilent)
Standard HPLC systems traditionally accommodate 4.6 to 2.1 mm ID columns with 3.5 to 5 micron packings. The typical maximum pressure of 400 bar (6000 psi) works with column length up to 250 mm. Columns such as Agilent’s Rapid Resolution High Throughput columns can be run at lengths up to 100 mm on HPLC instruments. However, a variety of sub-2-micron particle columns creates higher back pressures and requires UHPLC systems with a pressure range above 600 bar and a flow rate above 2 ml/min.

Carolus (Pharmacare)
HPLC is proven separation technique used for more than 30 years. UPLC/UHPLC is a new separation technique based on the fundamentals of HPLC. UPLC systems uses columns with sub 2 µm particle size and operates at much higher pressures than conventional HPLC.

HPLC Advantages:

  • been around for more than 30 yrs.
  • it can be used for a variety of applications.
  • various detection techniques.

HPLC Disadvantages:

  • run times are longer than UPLC. Run times can be as long as 60 minutes in some cases.
  • resolution/efficiency is much lower than UPLC
  • maximum pressure on normal HPLC’s is ±5000 psi

UPLC Advantages:

  • run times are shorter than HPLC. Therefore, increased lab throughput.
  • it uses the fundamentals of HPLC and takes separation science to the “next level”.
  • increased sensitivity .i.e. lower Limits of Detection than HPLC.
  • max pressure ± 15 000 psi
  • peak capacity is increased

UPLC Disadvantages:

  • Sample “cleanup” is critical to the column lifetime.
  • Running costs of the system is generally higher than HPLC running costs due to the higher back pressure of the system. This will be offset by the increased throughput.
  • Precautions must be taken to ensure that mobile phases are filtered through 0.22µm filters

McGinley (Phenomenex)
Many define a UHPLC system as a system that can operate at backpressures above 400 bar, which allows the use of sub-2µ particle HPLC columns. UHPLC instruments also tend to be a “tighter” system with fewer extracolumn volumes compared to older HPLC systems. The advantage these UHPLC systems provide is that the reduced extracolumn volume and higher backpressure limits of such systems allow for the improved performance that sub-2µ columns and new technology media provide. The major downside that many UHPLC methods have is that they are not backwards compatible (i.e. a method developed using a UHPLC system with a sub-2µ HPLC column cannot be transferred to existing HPLC systems).

Khanna (Waters)
UHPLC or other high pressure LC systems simply take HPLC to higher pressure limits and thus provide speed improvements, but potentially at the expense of data quality as dispersion and other factors may compromise the separation. UPLC, on the other hand, takes advantage of the ultra high pressures but is optimized for performance at those pressure levels. Therefore, the more important distinction for the high performance laboratory is the difference between UPLC and UHPLC.

What strategic business advantages does each offer?

McGinley (Phenomenex)
As HPLCs with higher backpressure limits (UHPLC) displace older HPLCs during the next 10-20 years, equipment with higher backpressure limits will allow more flexibility in HPLC method development. This is dependent upon such instruments being compatible with a majority of the separation device possibilities in the market and being able to integrate readily with all mass spectrometry solutions; global concerns shy away from vendor-specific solutions.

Khanna (Waters)
There are also numerous operational efficiencies of UPLC within high throughput operations that include increasing the productivity within the same floor space and reducing the environmental impact with less solvent consumption. At the workbench, we also have seen improved chromatography, even with less experienced technicians running the equipment.

How does a “one size fits all” approach apply for different laboratory testing needs? What are the advantages? Disadvantages?

Schulenberg-Schell (Agilent)
In contract labs, for example, we see rapidly changing requirements as new contracts come along and each customer has different demands. A setup that can be used for various projects and associated methodologies without redeveloping the methods delivers much more uptime and revenue-generating results. Time spent reconfiguring instruments becomes a nonissue.

McGinley (Phenomenex)
“One size fits all,” or laboratory standardization across instruments and columns used, has some advantages in improved redundancy and streamlined laboratory costs; however, this comes at a cost of flexibility and performance.

Khanna (Waters)
Relying on identical instruments to address the needs of varying laboratories ensures scientific mediocrity in which basic challenges are solved, but specific functional requirements and challenges are not being met. The other extreme is that every lab function relies on different technologies, compromising an organization’s continuity.

How can a pharma company quantify a  successful new or  enhanced LC technology implementation?

Schulenberg-Schell (Agilent)
One way is to measure throughput, for example, generating two to three times the amount of high quality results using the latest LC equipment and column technology versus conventional. Another is instrument utilization, for example, being able to replace aged instrumentation with half the number of instruments that get utilized more fully.

McGinley (Phenomenex)
Although quality of a method is hard to quantify, throughput improvements are actually easy to analyze. The number of samples processed in reduced time and money can easily be quantified.

Khanna (Waters)
For every customer, the business impact of UPLC will vary based on its application, from improving the drug discovery process to maximizing product release efficiency. For a CRO, it can mean the ability to take on more clients and meet or exceed delivery timetables while providing greater levels of client service. For generics, it can mean meeting delivery targets for retail customers potentially with first-to-file six months exclusivity.

Considering the current economic climate, what are the realistic expectations for ROI of this investment? What are the parallel benefits that are often overlooked?

Schulenberg-Schell (Agilent)
The increased separation efficiency per dollar and hour can be used to satisfy different critical business needs. It may be higher throughput enabling a lab to cope with higher sample loads, freeing up resources for other tasks. Second, the separation power is used to achieve a higher resolution.

Carolus (Pharmacare Limited)
Using new technology columns will have an immediate impact on the productivity of your lab and may not require revalidation of your analytical methods. This would probably be the easiest way to leverage the benefits these columns have to offer. Investing in a UPLC system would be in the region of $100,000 and would probably pay for itself after about two years. The column could be considered to be a consumable and is generally only a fraction of the total cost of the analysis of a sample. Therefore, if the column only lasts a few thousand injections, the benefits far outweigh the cost of a new column.

McGinley (Phenomenex)
Any technological improvement that can be implemented without investment in capital improvements is practical in the current climate. However, UHPLC capital expenditures for improved throughput are currently difficult to justify when alternatives exist. An added benefit that such methods provide that is often overlooked is reduced solvent costs. In some cases, an improvement in sensitivity can be observed, leading to improved results.

Khanna (Waters)
Some companies have increased sample throughput without having to invest in increasing the size of their laboratory workspace. We have also received feedback that, in some instances, the increased operational efficiencies of UPLC have actually paid for the initial cost of the system in a far shorter time span than typically expected, often justifying the investment. For example, solvent savings alone will often offset any capital costs. Companies are, in many cases, reinvesting the savings back into additional UPLC technology upgrades.

What future developments do you see for LC technology?

Schulenberg-Schell (Agilent)
Liquid chromatography is gaining popularity among more users in life science and engineering disciplines. These environments require new ease of use and additional sensitivity. LC and LC/MS are major tools for discovering the secrets of biological pathways. Innovations such as a phospho-peptide workflow based on a robust nano-spray HPLC-Chip/MS system are enabling biologists to enrich and analyze posttranslational modifications in a single step.

Carolus (Pharmacare)
From a hardware point of view, I think we will see detectors with increased sensitivity/sampling rates and reduced injection cycle times for autosamplers. I think there will be gradual improvements on the hardware as manufacturers try to stretch the boundaries of separation science. I think there will also be improvements in column chemistry (e.g. using columns with maybe 1.5 µm particle size columns).

McGinley (Phenomenex)
The line between HPLC and UHPLC will blur as all new HPLCs will have high pressure capabilities and reduced extracolumn volumes. As for column developments, one should expect to see further refinements in media morphology for increased separation power and further improvements in efficiency.