Expect Nothing Less: Expectations of a Contract Research Organization

By Jon Rhodes, M.S.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

The pharmaceutical, agrichemical, chemical, and biotechnology industries depend heavily on Contract Research Organizations (CRO’s) to provide high quality, scientifically sound laboratory and field-based services.  And to do so with minimal input from the Sponsoring organization on the routine aspects of study design, conduct, and reporting.  After all, many CRO’s have decades of cumulative experience relative to experimental design and execution as well as what regulators are likely to accept.  In addition CRO’s have deep knowledge of standard practices and individual registrant nuances that can be leveraged to ensure high quality and regulatory acceptability. The overriding goal is to ensure quality science while meeting challenging development timelines.

The basic expectations of a Contract Research Organization have not changed:

• The CRO will conduct studies that satisfy scientific regulatory requirements and customer reporting standards

• The CRO will ensure communication is proactive and timely

• The CRO will offer proactive technical advice and feedback based on practical experience

• The CRO will ensure consistency of approach across all projects within the organization

Likewise the basic expectations of a Registrant have not changed:

• The registrant will provide all study specific information relative to desired project design and will provide deadlines for deliverables

• The registrant will supply specific information about the test material (if available) including analytes and analytical methods, expected behavior of test material, expected toxicity, and any specific requirements required to ensure consistency of testing strategy

• The registrant will provide technical advice and feedback based on practical experience with the test material and experience of approaches received from regulators

• The registrant will ensure timely communication to the CRO relative to study plan and report reviews, responses to study updates and suggestions/questions, and timeline changes

A lot of what makes a successful partnership isn’t strictly science – communication and transparency are everything.

Radiolabeled CTMs in Early Phase Development under GLP

By Wayland Rushing, Ph.D.

Senior Scientific Advisor

ABC Laboratories

www.abclabs.com

Radiolabeled products are used extensively during pre-clinical studies in BA/DMPK studies. The material used for these studies is typically research grade material released under Good Laboratory Practices (GLP’s). However radiolabeled drugs are also used during ADME and bio-availability studies. Since the products are now intended for use in human studies, they now must comply with CGMP regulations in terms of its manufacture and release testing.

While the processes by which CGMP’s are applied to the synthesis, control and testing of non-labeled materials is well understood, we have found there is a fair amount of confusion in terms of the regulatory requirement and how to apply those to radiolabeled products.  Here I want to address one specific aspect of this: Analytical Methods and Testing.

Typically during the point in development that CGMP radiolabeled materials are needed, significant work has been performed on the unlabeled material.  This normally means that analytical methods and testing specifications have already been developed and validated accordingly.  There are two issues which may occur in implementing these analytical methods for testing of the labeled material:

·       Impurity profiles: The impurity profile of the radiolabeled material may be significantly different than the un-labeled material.  This may be the result of the synthetic route having to be altered to prepare the labeled material and/or  radio-induced degradation which can lead to new impurities being formed.  This requires evaluation of the existing methods to ensure that they still properly work for determination of the chemical purity without interferences.

·       Radio-purity determination: One of the key specifications of the labeled material is the radio-purity of the final material.  The existing analytical methods are not able to determine this as it requires the use of a radio-detector (typically in-line with HPLC).

Since the testing of the final material is required to be CGMP compliant then the methods used are required to be developed and phase-appropriately validated for their intended use.  As a result the analytical testing for radio-labeled materials can be a complex process requiring: method transfer (of existing methods), method development and phase appropriate method validation.  Hence it is critical that at the initiation of a CGMP radiolabeling program a thorough plan is designed and implemented to ensure that not only is the material synthesis accordingly to meet the regulatory needs, but also that the analytical methods are also appropriate for use for the testing and release of the final product.

I will expand on this topic at the 12^th International Symposium on the Synthesis and Application of Isotopically Labelled Compounds on the campus of Princeton University June 7-11.  Join me at 10:30 AM June 11 for a podium presentation titled, “CGMP Radiosynthesis for Early Clinical Trials: A Unique Challenge.”

New Frontiers in the Analysis of GMO Crop Proteins

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

Genetically Modified Organisms (GMO) have been on the market for over two decades. These plants have been engineered for a variety of properties including:

  • Herbicide resistance

  • Cold/heat tolerance

  • Disease resistance

  • Increased yield

  • Improved quality

  • Pest resistance

Either internally or through licensing agreements large Agro Science companies have combined many of these properties into a single species. This may result in the introduction of 10-20 modified proteins. Each protein is present at different levels in each plant tissue and these levels typically change within the lifespan of the crop. The public concern with GMO crops has led to stringent control. The licensing requirements require careful control of the plants (particularly seeds), multiple field trials and careful monitoring of the modified plant proteins.

This presents quite an analytical challenge: 10-20 modified proteins in up to 12 different plant tissues. Sample preparation alone presents a daunting task. Plants are separated into their component tissues and each tissue macerated (multi-step), often lyophilized and ground to a fine homogeneous powder. The proteins are extracted, typically requiring different extraction methods depending upon the particular protein or tissue.

Once prepared and extracted actual quantitation of the proteins is required. The technique must be sensitive (low ng/mL), specific (thousands of proteins) and precise (crops are sampled several times during their lifecycle). Based on these requirements the methods available are:

  • ELISA

  • Western

  • LC/MS/MS

ELISAs are currently the method of choice. As I discussed in a previous blog (“WES, an alternative to ELISA”, 4/15/15), ELISAs possess the sensitivity and specificity required for GMOs, but not the day-to-day precision and are labor intensive. Automated Western analysis, as performed with the Wes™ system (Protein Simple®), alleviates many of the issues of with ELISA. It shows excellent day-to-day precision and is highly automated. However, both of these methods have relatively low sample throughput, 25-35 samples per plate with total analysis time from 2.5 – 18 hours. Given the hundreds of samples generated for a single GMO field trial, each of which require analysis of 10-20 different proteins, these techniques require man-months of analysis time. While ELISA can be multiplexed, this too is a laborious process and is better suited for analysis of a single matrix (plasma).

Within the last few years the use of proteomics, specifically LC/MRM/MS, has appeared in the literature for the analysis of GMO proteins. This technique appears to possess all the requirements necessary including high throughput. To provide the specificity required the mass/charge ratio must be determined for each of the proteins of interest. Typically the necessary sensitivity cannot be obtained analyzing intact proteins; therefore, proteolytic peptides are utilized. The entire plant extract is proteolytically digested (e.g. trypsin, Lys-C, etc.). This mixture of thousands of peptides is then analyzed by UPLC/MS/MS. Most of the peptides co-elute with multiple other peptides, but through the use of powerful proteomic software the peptides of interest can be teased out of the background. The chromatographic resolution is then optimized and the use of MRM (Multi Reaction Monitoring) is incorporated. In MRM, the peptide ion is separated by its mass/charge ratio in Q1, reacted in Q2 to produce daughter ions fragments which are further separated in Q3. MRM increases the sensitivity of the method 100-fold and provides an additional level of specificity by monitoring two daughter ions. Optimal precision is obtained through use of internal standards, usually synthetic ¹³C-labeled daughter ions. While this analysis requires substantial development time (as does ELISA), once developed it is a highly automated (walk-away). Its ultimate advantage is that MS/MRM can be multiplexed; there are reports of twelve or more proteins quantitated in a single analysis. This results in sample throughput 5-10 fold greater than ELISA or Western.

In summary, MRM/MS has the following advantages:

  • Accurate

  • Highly specific – two daughter ions

  • No requirement for antibodies

  • High throughput/multiplexing

  • Highly automated

While currently not the method of choice, MRM/MS seems poised to make enormous inroads for the analysis of protein levels in GMO plants.