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Alternative Approaches for Determining Protein-Ligand Binding Constants

An alternative approach to label-free determination of protein binding strengths with small molecular ligands is Surface Plasmon Resonance (SPR).  SPR is a method for characterizing macromolecular interactions.  It is an optical technique that uses the evanescent wave phenomenon to measure changes in the refractive index very close to a sensor surface.  The binding between an analyte in solution with a ligand on the sensor surface results in a change in the refractive index.  The interaction is monitored in real time and the amount of bound ligand and rates of association and dissociation can be measured with high precision.  Analysis of kinetics and thermodynamics by SPR can be used to understand the complex mechanisms of molecular recognition events.  The great advantage of SPR is the sensitivity and easy access to kinetic data of the non-covalent binding processes.

However, a disadvantage for conventionally SPR-based measurement is that no stoichiometric information is available, and that the process of immobilization is sometimes delicate and often demands lengthy optimization.  It is crucial to minimize or to completely avoid non-specific surface–analyte interactions during the binding experiments.  The sensitivity of an SPR instrument can be a problem since the angle of reflection is detected which is proportional to the mass of the analytes and can generate poor signals for small molecules.  Other disadvantages of SPR have been described in the research article "Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry" written by Matthias C. Jecklin, Stefan Schauer, Christoph E. Dumelin and Renato Zenobi.

Approaches to Determining Protein-Ligand Binding Constants

The non-covalent binding of small molecules, ligands to proteins, is playing a crucial role in biopharmaceutical research.  This interaction would alter the stereochemistry of a protein molecule (a drug candidate) and also could modify its molecular recognition ability and ultimately its bioactivity.  Therefore, it is important to develop a suitable method which is able to quantitatively determinate the binding strengths of the molecule.  A variety of different approaches have been developed to quantify molecular interactions.  Methods applied to drug-protein binding studies in the pharmaceutical and biomedical sciences include equilibrium dialysis, ultra-filtration, ultra-centrifugation, gel filtration, calorimetry, microdialysis, spectroscopic, HPLC, and capillary electrophoresis-based methods.

The methods that are traditionally used for measuring the binding strengths (e.g., fluorescence polarization or radioassays) require labeling of the analytes that cause problems related ether with complicated synthesis, generates radioactive waste, or produces incorrect results.

Normally, interaction between a protein and a ligand shows the dependence of pH, reflecting the linkage between the binding of the ligand and the binding of protons.  This linkage is quantitated as a change in the ligand binding constant with pH, or as a change in the proton affinity (i.e., pKa) of an ionizable group in the protein upon ligand binding.  In determining proton linkage, one typically measures the affinity constant for the binding of the ligand at a number of pH values and calculates the pKa of the protein in the free and liganded states.  However, this is problematic if the ligand binding constant is too large to be readily measured or if the pKa shift is small.

A common, truly label-free solution for measurement is Isothermal Titration Calorimetry (ITC).

ITC is a thermo-dynamic technique that allows the study of the interactions of two species.  When these two species interact, heat is either generated or absorbed.  By measuring these interaction heats, binding constants (K), reaction stoichiometry (n) and thermodynamic parameters including enthalpy (∆ H) and entropy (∆ S), can be accurately determined.  In addition, varying the temperature of the experiment allows the determination of the heat capacity (∆ C_p) for the reaction.

Titration experiments are typically fast (approximately 1 hour) yielding accurate values of K (in the range of 10² to 10⁸ M^-1), n, ∆ H and ∆ S.  No labeling or immobilization is required.  Also, ITC is not limited by the ligand or protein size.  It is relatively artifacts-free and is not affected by the optical properties of the samples.  ITC allows researchers to study almost any kind of interaction, including solutes with immobilized enzymes, tissue samples, or other solid materials in suspension.  The only major disadvantage of ITC is that it requires relatively high concentrations of samples.

In Vitro Testing Models for Nasal Drug Delivery

Nasal drug delivery is a very promising alternative route for various drugs included hormones, vaccines, peptides, proteins or other large macromolecules.  Nasal drug delivery has also been generating widespread interest in the drug delivery field because it could not only be used in local treatment but in systematic administration.  Therefore, evaluation of these novel formulations requires reliable in vivo/in vitro testing models.  The nasal mucosa provides a moist and highly vascularized membrane, crucial to rapid absorption into the blood stream, thus facilitating faster transport to the site of action.

Compounds administrated via this route are absorbed directly into the circulation system, avoiding the first-pass hepatic metabolism.  This site of drug administration has been considered an ideal route for non-invasive delivery route.  However, there are a number of factors that limits the widespread utility of this route.  The two main disadvantages of nasal delivery are the limited maximum dose per spray and the rapidity of clearance from the nasal cavity, enzymatic degradation in the mucus layer and nasal epithelium.

Various factors that might affect the permeability of drugs through the nasal mucosa could be broadly classified into three categories:

1. Biological factors

2. Formulation aspects, and

3. Device-related factors

The physiology of the nasal cavity presents the most significant barrier to drug absorption.  However, the problem associated with low bioavailability has been solved recently by developing an inter-nasal micro-emulsion formulation delivery system.  Hence, a formulation that would increase residence time in the nasal cavity and at the same time increase absorption of a drug would be highly beneficial in all respects.  The use of bioadhesive polymers has been shown to lengthen the residence time and enhance the bioavailability of drugs delivered to the nasal cavity.

Excised animal tissue models are frequently used for nasal drug absorption studies due to methodological and ethical limitations associated with the use of human nasal specimens, although it is difficult enough to obtain human nasal tissue without these other issues.  The fresh nasal tissue removed from the nasal cavity of sheep is commonly used for this purpose.  The cumulative amounts of drug permeated within determination of the effective permeability coefficients across mucosal membrane are calculated.  The histology of treated nasal mucosa membranes also has to be investigated after the completion of the experiments.  The porcine nasal mucosa also seems to be feasible for in vitro studies that investigate the permeability of nasal tissue and predict the absorption of drugs after nasal administration in vivo.

Mainly there are three in vitro testing models:

1. Excised models: excised animal nasal mucosal tissue is obtained from various animals that are frequently used to study nasal transport and metabolism (rabbits, bovine, sheep tissue).  It is crucial to select the right part of the nasal mucosa tissue for in vitro investigation studies.  The different regions of ovine nasal mucosa cavity demonstrate quite different in vitro drug permeability properties.  It has been established that the middle turbinate mucosa is the suitable model for in vitro studies because of its many advantages such as large surface area, highest drug permeability coefficient and high reproducibility.

2. Cell line models: RPMI 2650 human nasal epithelial cell line derived from a spontaneously formed tumor.  These include primary and passaged cell culture, Liquid-Covered Culture method (LCC) and Air-Interfaced Culture method (AIC).  Cultures of human nasal epithelial cell layers on Transwell inserted under AIC or LCC conditions are useful for in vitro drug transport studies.  MatTek Corp. (200 Homer Avenue, Ashland, MA) offers EPI tissue models.  However it must be noted that efforts to develop and characterize various nasal cell culture systems are still in their infancy.

3. Several artificial membranes have also been proven to be a good model for nasal drug delivery studies.  For instance, the polydimethylsiloxan (PDMS) membranes have demonstrated good in vitro model properties during nasal mucosa drug permeation studies.

Qualitative and Quantitative Aspects of Peptide Mapping Using UPLC-UV

As outlined in my previous post, there are several critical factors that must be considered to validate a method used for peptide mapping, and each of the factors, along with the acceptance criteria, should be designed into a protocol or Standard Operating Procedure (SOP).  Because there is a wealth of general validation guidance available, discussion will be restricted here to areas where peptide mapping validation might differ from other types of methods (for example, methods for synthetic drugs).

Robustness

To evaluate a peptide map against a standard, the chromatographic separation must be robust.  Although general chromatographic robustness has to be applied however, there are additional issues to consider in a peptide map method, and these include (enzyme) reagent quality or purity and digest stability.

Column considerations also must be made during a proper robustness study, since it is a well known fact that no two chromatographic columns are created equal.  When determining the robustness of the reagents used for digestion, it is common to evaluate a protein reference standard of known composition with cleavage agents from different lots.  The number of peaks obtained, their shape, and the peak areas all are compared in the resulting chromatograms.  Because in some cases chromatographic run times can by quite long, the length of time and the conditions under which a digest can be stored before being analyzed also must be evaluated as part of a robustness study.  Digest stability usually is evaluated by looking for significant differences in the map resulting from the analysis of several aliquots of a single digest stored at different conditions.  It also can be desirable to investigate stability through to several freeze-thaw cycles.

Lastly, it is a well known fact that no two chromatographic UPLC columns are created equal.  Although the manufacturer Waters today provides much better control of their processes than in the past, minor column differences can have a significant effect on the separation of these complex samples.  It is a good idea to evaluate the reference standard on several different UPLC systems, column lots, and evaluate column life, because as a column ages, the separation can be affected.

Linearity and Limit of Detection

The serial dilution of a peptide mixture should be made in order to demonstrate that there is no significant shift in retention or deterioration in peak shape from low to high levels.  It has to be confirmed that the dynamic range of the chromatographic material and gradient method is sufficient for the analysis of a small amount of one peptide in the presence of much larger amount of another.  The detector response should be linear with the sample amount.

Six replicate injections should be overlaid to demonstrate the reproducibility of UPLC-UV peptide mapping method.  The same sample might subsequently be injected at different levels to test linearity and sensitivity.

The limit of detection (LOD) in a peptide map is determined by the ability of the method to distinguish changes in the map, for example, the presence or absence of a peak.  Experiments can be carried out to modify the target protein intentionally and then a digest of the modified protein is mixed with a control digest or standard reference material in varying proportions.  Ideally, a decrease in peak response for the unmodified peptide and a corresponding increase for the modified peptide should be observed.  Peptides modified by oxidation, deamidation, glycosylation or other mutations usually have reported LOD's in the range of 2-15 mol% .  The sensitivity in combination with robust chromatographic behavior enables detection of low level peptides in a complex digest.

Precision

Precision in peptide mapping is measured on two levels; repeatability and reproducibility from both intra and inter-testing runs. Repeatability is measured by running six replicate injections of a single pooled digest of the reference standard.  When repeatability is performed in this manner, all variability from the sample and reagents are eliminated, and the true instrument or system component of precision can be measured and used to help set system suitability criteria.

Intra and inter measurements are the more important parameters to be evaluated during validation, however. Intra-test precision is the reproducibility of the fragmentation (digestion) and the chromatographic separation.  Acceptable precision is obtained when the peak retention times and areas are constant from chromatograms obtained from consecutive tests of a series of separately prepared digests of the test protein.  The average standard deviation of the retention times and areas should not exceed a pre-determined specified acceptance criterion.

Inter-test precision is what traditionally has been referred to as intermediate precision or true reproducibility.  It is a measure of the reproducibility of the peptide map when the analysis is run according to an experimental design made to measure the effects of the test run on different days, by different analysts, in different laboratories on different systems, different column lots.  For inter-test precision, the experimental design should include comparisons using peak retention times and areas relative to an internal standard peak within the same chromatogram.  By using relative values, the need to make adjustments for things like injection volume differences, column volumes and instrument gradient delay volumes is eliminated.

In general, it can be expected that %RSD for peak retention times and areas will be greater for the inter-test compared with the intra-test precision, which in turn will be greater than the repeatability results.

System Suitability

The guidance on system suitability can be found in the USP chapter on chromatography.  Like any other method, the acceptance criteria for system suitability of a peptide map depends upon the identification of the critical test parameters that affect data interpretation and acceptance.  System suitability limits, for both recovery and chromatography, are determined by running a reference standard in parallel with the test protein and looking for indicators that monitor, for example, that the desired endpoint was reached in the digestion; normally, selectivity and precision.  However, the consistency of the pattern obtained is best defined by peak-to-peak resolution.  Additional chromatographic parameters such as peak width, tailing factors and column efficiency also can be used.

The parallel study (reference standard and test protein) also is used to visually compare each peaks relative retention time, responses (peak retention time and area), the number of peaks and the overall elution pattern.  This comparison often is complemented by mixing the two samples (1:1, v/v) and evaluating the peak response ratios and elution pattern.  If all peaks in this mixed sample have the same relative retention times and peak response ratios, then the identity of the protein test sample can be confirmed.  Significantly different retention times are also an indication of system variability, while the appearance of new or broader peaks indicates non-equivalence.  Computer-aided pattern recognition software and other automated approaches have been used on occasion to examine the degree of difference or similarity when comparing two different peptide maps, but these have not gained routine acceptance.

Conclusion

Although most of the underlying principles still apply, the validation of a peptide map includes some additional considerations when it comes to LOD, robustness, and precision, and depends upon the stage of the regulatory process.  While mostly involving comparative testing, when properly validated, a peptide map can be used to accomplish its intended purposes: to confirm the primary structure of a protein; to detect whether or not alterations have occurred; and to demonstrate process consistency.

Peptide Mapping Using UPLC-UV Method Development Techniques

Peptide mapping or peptide finger printing produced is characteristic for a particular protein and the UPLC technique can be used to separate a mixture of peptides.

Normally, recombinant proteins are developed for therapeutic purposes.  Peptide mapping is used to confirm the primary structure of a protein, identify post-translational modification, to demonstrate generic stability and analyze potential impurities.  Any difference in the structure of a protein should be reflected in a change in retention time for the peptide containing the modification.  The relative amounts of the peptide with and without a particular modification are used to measure the fraction of the protein in the particular sample that carries that modification.  Changes in area proportions correspond to the fraction of the protein molecules in the sample having a particular modification.  Using a UPLC technique, peptide analysis has been shown to give consistent chromatographic separations and reproducible quantitation for peptide mapping in combination with UV, MS or MS/MS detection.

In the initial characterization of a protein, it's important to develop a peptide mapping method that resolves modified peptides from native peptides so that all possible modifications may be detected. As development of the biopharmaceutical advances, these peptides must be quantitated.  Quantitation is generally expressed as an area or height percent of the native peptides.  In this way, the peptide map can provide information on the mixture of protein forms in each sample so that safety and efficacy of the preparation may be assured.  The method must, therefore, exhibit excellent sensitivity and linearity for quantitative work.

The strategy of peptide quantification includes adding a known amount of a specific peptide (peptide standards) to an actual protein digest to test estimates of quantification.  This peptide serves as a surrogate, illustrating the behavior of modified peptides in the digest.

Method validation Viewpoint

At the Investigational New Drug (IND) phase, limited validation is necessary; typically only an approved test procedure that includes system suitability as a test control.  Sometimes termed qualification, complete characterization of the individual peaks is not needed.  As the regulatory process proceeds, a partial validation might be needed to give assurance that the method performs as intended in the development of a map for the test protein.  However, validation of peptide mapping in support of further regulatory submissions requires a rigorous characterization of each of the individual peaks in the map.  Methods that are used to characterize the peaks in a map commonly use mass spectrometry (MS).

There are several critical factors that must be considered to validate a method used for peptide mapping, and each of the factors, along with the acceptance criteria, should be designed into a protocol or Standard Operating Procedure (SOP).

The critical factors include robustness, the limit of detection, specificity, linearity, range, accuracy, and precision. Recovery and reagent stability are also important to consider during method validation.  Recovery can be addressed by performing either quantitative amino acid analysis, spiking studies, or radiolabeling.  Many of the validation parameters must not only address the separation, but also the fragmentation or digestion, particularly when considering robustness studies.  The protocol also should include written test procedures that give a detailed description of the analytical method.

USP Advisory Panel Re-issues Draft USP General Chapters on Topical & Transdermal Drug Products Dissolution Testing into PF 35(3)

Today, I would like to draw your attention to a couple of recent changes to the USP Dissolution Testing monograph that you may need to be aware of.  Two new draft USP General Chapters on Topical and Transdermal Drug products have been published in the Pharmacopeial Forum Vol. 35, No. (3) May-June 2009.  The General Chapters are: <3> Topical and Transdermal Products-Product Quality Tests; and <725> Topical and Transdermal Products-Product Performance Tests.  You can find these if you follow the links provided above.

These are important developments since dissolution testing is plying a very important role in the pharmaceutical industry during drug development, quality control and stability programs.  The test is used in order to assure consistent product (batch) quality within a defined set of specification criteria and all products must pass to be on the market.  Initially, the dissolution testing procedure was developed for immediate release solid oral dosage form products and later it was extended for extended/controlled/modified release solid oral dosage form products.  Recently, the application of dissolution testing has widened to a variety of "novel" or "special" dosage forms, such as suspensions, orally disintegrating tablets, chewable tablets, chewing gums, transdermal patches, semisolid topical preparations, suppositories, implants, injectable micro-particulate formulations, and liposomes.  It is referred to as the "drug release test".  Until now, because of significant differences in formulation design among these novel/special dosage forms, it was not possible to develop a single test protocol that could be used to study the drug release properties of all products.  Rather, different apparatus, procedures, and techniques have been employed on a case-by-case basis.  The two new draft USP General Chapters on Topical and Transdermal Drug products have been published in Pharmacopeial Forum 35(3) May-June 2009 to address this need.

These two General Chapters are part of a series of chapters that will cover product quality and performance tests for the five routes of administration.  In addition to the transdermal route, the other routes of administration include injection, mucosal, inhalation, and gastrointestinal.  The Draft General Chapter <3> is the first in the default monograph series.  For oral dosage forms (gastrointestinal), <711> and Dissolution, <724> Drug Release are examples of product performance chapters.

The general Chapter <725> covers the apparatus and procedures used to evaluate the in vitro drug release and proposes a performance verification test to assess equipment performance.  The product performance test is consistent with that proposed in the FDA Guidance for Industry, Nonsterile Semisolid Dosage Forms, Scale-up and Postapproval Changes: Chemistry, Manufacturing and Controls; In Vitro Release Testing and In Vitro Bioequivalence Documentation (SUPAC-SS).

All this information has been posted on the USP web site.  Comments and suggestions for these tests and procedures are invited from interested parties through the routine Pharmacopeial Forum comment process.  I encourage everyone who may be affected by this to participate.

Dissolution Testing Methods for Poorly Soluble Compounds - continued

As I pondered my last blog entry, I felt that I needed to expand on the topic of dissolution methods for poorly soluble compounds for sustained or extended release formulas.

Time release technology, also known as sustained-release, extended-release, controlled-release, modified release or continuous-release is a mechanism used in tablets or capsules to dissolve slowly and release a drug over time.  The advantages of sustained-release tablets or capsules are that they can often be taken less frequently than instant-release formulations of the same drug and that they keep steadier levels of the drug in the blood stream.

They involve complicated excipients and preparation technologies.  Sustained release formulations often need two kinds of dissolution media to simulate the human gastrointestinal pH-acid stage and buffer stage.  Some drugs are enclosed in polymer-based tablets with a laser-drilled hole on one side and a porous membrane on the other side.  Stomach acids push through the porous membrane, thereby pushing the drug out through the laser-drilled hole.  In time, the entire drug dose releases into the system while the polymer container remains intact, to be later excreted through normal digestion.

In some sustained release formulations, the drug dissolves into the matrix and the matrix physically swells to form a gel, allowing the drug to exit through the gel's outer surface.  The difference between controlled-release and sustained-release is that controlled-release is a perfectly zero-order release; that is, the drug releases over time irrespective of concentration.  Sustained-release implies slow release of the drug over a time period and it may or may not be a controlled-release.  Traditionally, a drug dissolution release test for these formulations could require a day to complete sample analyses, making dissolution testing a time-consuming and labor-intensive procedure.

Over the last ten years, traditional methods of drug dissolution testing have been gradually replaced by fiber-optic dissolution systems in which a fiber-optic probe is inserted directly into each vessel to perform measurements in situ.  Real-time drug dissolution release is determined in the vessels without sample removal, greatly simplifying the testing procedure.

The in situ fiber-optic dissolution system has proved to be productive, useful, labor saving and can acquire more information than conventional dissolution testing methods.  The rapid in situ data collection capability of the system makes it possible to characterize a real-time dissolution profile.  The fiber-optic dissolution system is widely applying for formulation development, bioequivalence research, and product release or stability studies.

Developing Dissolution Testing Methods for Poorly Soluble Compounds

Since the therapeutic effectiveness of a drug depends upon the bioavailability and ultimately upon the solubility of the molecule, the proper dissolution testing method plays crucial role in the new drug development process and quality control.

Currently only about 10% of new drug candidates have both high solubility and permeability.  Therefore, one of the current challenges in pharmaceutical product development is dealing with low solubility and/or low permeability.  A common strategy here is to go to smaller particle sizes, thereby increasing the specific surface area (SSA) and solubility of the product.  For very small particles with a nano or submicron size, either a bottom up or a top down approach can be applied. Other considerable factors that will affect drug solubility are polarity and polymorphism.  Generally non-polar drug molecules will dissolve in non-polar solvents and polar molecules will dissolve in polar solvents.  The different types of intermolecular forces such as Dipole-Dipole, London Dispersion or Vander-Waals have to be taken in consideration at early stage of drug development during the solubility studies.

Polymorphism is the ability of a compound to crystallize in different forms.  The two polymorphs cannot be converted from one another without undergoing a phase transition.  Polymorphs can vary in melting point.  Since the melting point of the solid is related to solubility, so polymorphs will have different solubility.

The medium, temperature and stirring are the factors that control a dissolution process.  In some cases, the method used in the early phase of product/formulation development could be different from the final test procedure used for control of the product quality. Indeed, methods used for formulation screening or understanding of the release mechanism may simply not be viable for quality control or drug stability studies.

During pharmaceutical product development, dissolution testing is primarily used in measuring the rate of drug release and solubilization, assessing the stability of the formulations, monitoring product consistency, assessing formulation changes, and establishing in-vitro in-vivo correlation (IVIVC).  For a commercial product, dissolution testing is primarily used to confirm product consistency, to evaluate the quality of the product during its shelf life, and to assess post approval changes and the need for bioequivalency studies.  It is essential that with the accumulation of experience, the early method be critically re-evaluated and potentially simplified, giving preference to compendial apparatus.

A novel in-vitro dissolution model based on the principle of flow-through technique has been recently designed in order to evaluate the in-vitro release rate of poorly water-soluble compounds.  The flow through apparatus (USP 4) has been coupled with the compendial dissolution apparatus (USP 2).  The main objective of this study was to develop a novel in vitro dissolution method that can be applicable to identify changes in formulation, thus avoiding expensive in vitro studies.

The existing compendial methods for poorly soluble drugs usually require huge volumes of dissolution media for complete release of a drug from its formulations. For example, a drug with a solubility of 1μg/mL and dosage strength of 25-mg will need 25 liters of dissolution medium for complete solubilization.  This makes the dissolution testing physiologically irrelevant and practically difficult.  The in vitro release profiles obtained from this dissolution model were able to distinguish the formulation changes of several poorly water-soluble drugs from their dosage forms.

The model has successfully demonstrated bioequivalency and/or non-bioequivalency for commercial formulations.  In the future, an established IVIVC model can be used to characterize the biopharmaceutical quality of products at different stages of formulation development.

When the opposite is true, where dissolution is "too rapid", conventional dissolution approaches won't work here and either way, a customized in vitro dissolution method needs to be developed where a stable and reproducible method can be validated.  This requires more research to be conducted.  So, solubilization of poorly soluble drugs is a common problem.  Rapid dissolution should be fine for immediate release formulations unless one is attempting to reduce solubility on purpose to have more control over the dissolution rates that could eventually alter drug absorption in pre-determined ways.

Adapting New Analytical Technologies with Scientific Expertise

Implementation of new analytical technologies plays a crucial role in designing and controlling manufacturing processes for raw, in-process materials and final product quality. Introduction of new analytical initiatives helps build quality into the product and manufacturing processes, as well as continuous process improvement.

As Charles Darwin told us, it is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.  Both the FDA and industry experts expect benefits over conventional manufacturing practices: higher final product quality, increased production efficiency, decreased operating costs and better process capacity. Correspondingly, fundamental Changes are also expected within the analytical services CRO's which have to modernize their analytical capabilities, scientific expertise and compliance/regulatory standards. The future of pharmaceutical production will require innovative technological approaches and more science-based processes. Introduction of a new generation of analytical technologies will boost collaboration between research and development and manufacturing departments both inside companies as well as with analytical CRO's and increase overall efficiency. Approvals and inspections will increasingly focus on scientific and engineering principles. As a result, regulators will set higher expectations for new products from the outset.

Pharmaceutical companies are facing growing demands for increased productivity and reduced manufacturing costs. They also have to meet the evolving need for higher quality standards and higher drug expectations. Therefore, the role of independent analytical experts and CRO's equipped with the new technology is significantly increasing. The cost factors (affordability) and the higher scientific throughput of the new technologies that have recently been introduced dictate that manufacturers build closer collaborations with specialized analytical CRO's.

In recent years, a trend of change has been observed within pharmaceutical industry. As modern drug discovery has reached a remarkable level of complexity and drugs need to be discovered, developed and produced against strict timelines and within cost and regulatory constraints, industry seeks "lean"solutions to increase productivity. Among them, increasing the sample throughput of the ever-growing number of necessary (routine) analyses has become a popular target to cut costly time.

For the last thirty years, High-Performance Liquid Chromatography (HPLC) has been the leading technology when it comes to various analyses in the pharmaceutical industry.  However, the necessity of serial analyses taking typically 10-45 minutes has been a sample throughput-limiting barrier. Lately, the fundamentals of HPLC have been exploited to raise new technologies that can speed up analyses to ground breaking limits, without compromising separation efficiency. The Ultra-Performance Liquid column with 1.7μm particle size handling pressures up to 15000 psi and High Temperature Liquid Chromatography (HTLC) have the potential to take liquid chromatography to the next level in pharmaceutical industry. As each analytical method has its own demands, the advances of the above technologies has different applications in pharmaceutical analysis where high-throughput analysis can be meaningful, as in drug discovery, development and in quality operations. Both chemical and biological pharmaceuticals have considered the perspectives of these technologies and their realizations up to now in high-throughput pharmaceutical analysis.

Since its introduction to the pharmaceutical industry, liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) has played an important role in pharmacokinetics and metabolism studies at various drug development stages. Newly introduced techniques such as Ultra-Performance Liquid Chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques.

Accurate quantification of pharmaceuticals in biological fluids facilitates the correct determination of the pharmacokinetics of a medicine. Low-systemic-exposure compounds such as inhaled products or those undergoing extensive metabolism require very high sensitivity assays to accurately define the elimination phase of the pharmacokinetics curves. This need challenges the sensitivity of modern LC/MS/MS instrumentation.

The recently lunched Xevo TQ-S is an ultra-high-sensitivity tandem quadrupole mass spectrometer. It is equipped with StepWave optics featuring a revolutionary off-axis ion source design. The design of this source significantly increases the efficiency of ion transfer from the source to the quadrupole analyzer while the off-axis ion path eliminates neutral contaminants. These two factors combine to dramatically increase the sensitivity of the LC/MS/MS system.  Also, very valuable results have obtained by capillary electrophoresis and MALDI-TOF mass spectrometry for GLP studies of macromolecules in biological matrices.

UPLC Throughput Improvements Offer Much Faster Turnaround Times

Like most analytical CRO's, Diteba relies upon their High Performance Liquid Chromatography (HPLC) systems as being the workhorse of the laboratory.  From assay and purity work on raw materials, to stability sample testing, to bioanalytical analyses, the HPLC instruments are often critical to Diteba's and our sponsor's success.

When we initially equipped the laboratory, a significant evaluation was undertaken with respect to current HPLC technology.  One of the goals was to evaluate the latest technology on the market and how that could enhance laboratory productivity, improve assay resolution and sensitivity while still remaining to be robust and flexible technology.  With those goals in mind, Diteba invested in several Waters Corp.^TM AcQuity Ultra Performance Liquid Chromatography (UPLC) systems.

Although the concept of "Microbore LC" is not new, making it a practical reality was a challenge.  The AcQuity UPLC technology offers a new category of analytical separation science while maintaining the practicality of HPLC technology.  AcQuity UPLC systems are designed to reduce run times ten fold, while greatly improving resolution and sensitivity.  The technology combines AcQuity UPLC column chemistries with ultra-low dwell volumes, very high pressure pumping systems and low dispersion, high speed detectors.

The result is a very significant improvement in throughput, sensitivity and resolution. Quoting Dr. Steve Li, Diteba's Laboratory Operations Manager: "We find the UPLC systems to be equivalent to 3 or 4 regular HPLC systems in terms of productivity.  The resolution and sensitivity improvements have also allowed us to place many projects on UPLC, which would not have been possible with normal HPLC systems.  Having now placed different projects on the UPLC systems, including pre-clinical and clinical bioanalytical projects, the AcQuity column chemistries also seem to be more robust and forgiving".

The resulting improvement in throughput now means better turnaround times for Diteba's sponsors.  The more recent enhancement to Diteba's UPLC capability has been adopting a wider variety of detectors and interfacing with MS/MS.

Example of Turbinafene HCl Runs on HPLC and on UPLC:

In order to demonstrate improvements in UPLC technology, we prepared test runs on both a UPLC system and HPLC system using a Turbinafene HCl stability-indicating assay.  The comparison below from the Turbinafene HCl assay shows a resulting improvement in speed of greater than six times and sensitivity of almost 2.5 times of the UPLC system over the HPLC system.

If you have had similar experiences in evaluating laboratory equipment, we'd like to hear from you.