Proteomics and Validation

PFA-Proteomics Contract Research Services

The Proteome Factory is specialised in contract research for differential proteome analysis (proteomics, differential protein display) studies, identification and validation of regulated proteins, biomarkers and targets from all kind of protein containing samples. Only proteome analysis gives new insights in quantitative regulation of proteins caused by internal and external factors like diseases and drugs. The identification of regulated protein species (name, amount and modification) is the prerequisite to understand functional protein networks and their correlations. Biomarkers, biomarker profiles and targets can be identified. The proteomics information can be used to get better insights in in-vivo and in-vitro pharmacology or for optimization of fermentation processes and plant breeding.
For this purpose Proteome Factory has developed a comprehensive PFA-Proteomics Platform which opens new opportunities in biomarker and protein target discovery, quantification, identification and validation as well as pharmacokinetics of protein drugs. The proteomics platform consists of sophisticated gel-based and gel-free proteomics methods like iTRAQ™* proteomics and proprietary proteomics technologies ile MeCAT. In each proteomics study Proteome Factory decides in close cooperation with its partners and clients which proteomics technologies of the PFA-Proteomics Platform should be used to meet the scientific requirements. The study design, optimized sampling of biological material, sample prefractionation (e.g. affinity depletion of abundant plasma/CSF proteins) as well as statistical requirements are very important to ensure a high quality and valuable proteome study.
Picture: 2DE proteomics -

Application Areas of the PFA-Proteomics Platform

• Biomarker discovery and identification
• Biomarker profile discovery and identification
• Protein and peptide target discovery and identification
• Identification of regulated proteins and protein species
• Optimization of fermentation processes
• Optimization of plant breeding
• Pharmaco proteomics
• Identification of vaccine candidates
• Immuo proteomics
• Phospho proteomics
• Membrane proteomics

PFA-Proteomics Plattform

Proteome Factory performs gel-based and gel-free proteomics methods, which provide complementary information about the proteome.

• Biomarker- and Target Finding by extreme high resolution 2DE proteomics
• MudPIT (LC-ESI-MSMS based multi-dimensional protein identification technique)
• Label-free LC-ESI-MSMS proteomics
• ITRAQ based LC-ESI-MSMS proteomics
• Plasma Proteomics
• Immuno Proteomics
• Pharmako Proteomics
• Membrane Proteomics
• Pharmacokinetics by MeCAT (absolute protein quantification down to attomol range)
• Validation of biomarkers and target proteins by SRM / MRM analysis (mass western)
• Validation of biomarkers and targets by 2DE western blot and 1D western blot

Extreme High Resolution 2D Electrophoresis

Proteome Factory’s extreme high resolution two dimensional gel electrophoresis (2DE) based on the 2DE development of Professor Klose (Klose and Kobalz, Electrophoresis, 1995, 16, 1034-59) is still the most powerful separation technique for protein species worldwide allowing the best and most reliable proteome analysis. Several thousand protein spots (theoretically up to 10,000 protein spots depending on sample and gel size, up to 40x30 cm) can be separated and relatively quantified.
Even Proteome Factory’s 2DE gels with a size of 23x30 cm are much more powerful in protein separation than commercially available IPG-based 2DE because Proteome Factory’s 2DE technique has several advantages:
1. significant better protein separation resolution,
2. higher sensitivity with less protein amount,
3. less artificial streaks,
4. separation of basic protein up to pH 10.5 (up to pH 11.5 in 60x30 cm gels) and
5. high compatibility to protein identification by MS.

Workflow of Extreme High Resolution 2DE

The 2DE proteomics platform of Proteome Factory is very robust and highly standardized allowing reproducible analysis of all kind of protein containing samples using SOPs (standard operating procedures) without the need of time and cost intensive method development. Depending on scientific problem and customer request the 2DE procedure can be specifically optimized if required.
Proteome Factory’s 2DE approach comprises the following steps:

• Protein extraction
• Protein concentration determination
• 2DE protein separation (high resolution IEF and SDS-PAGE)
• Protein gel staining (MS compatible silver staining, Fluorescent staining)
• Digitalisation of 2DE gels
• Differential 2DE gel image analysis with differential protein display
• Evaluation of regulated protein spots with intensities, regulation factors and statistics
• Spot picking of differential protein spots by spotXpress
• Automated protein identification analysis
• Protein identification by database searching or de novo peptide sequencing

Advantages for Our Customer

• High quality and challenging proteome analysis for research & development
• Analysis results belong to customer
• Confidentiality
• Close collaboration
• Pilot studies to multi-year research projects
• High sensitive protein identification and quantification
• Highly qualified experts for protein and proteome analysis at Proteome Factory

*iTRAQ™ is a trademark of Applera Corporation or its subsidiaries.

The Importance of Clean Analytical Instrumentation Systems in Pharmaceutical Process Validation

by Dave Simko, Swagelok (Full-length paper presented at Interphex) Validated pharmaceutical processes are necessary to ensure quality of the products being manufactured, establish and maintain effective costs, and meet the regulatory requirements that are mandatory for approval and introduction of new pharmaceutical products. The collection of data needed to document the effectiveness and repeatability of the pharmaceutical process requires the use of a variety of both process control and analytical chemistry instrumentation. Process control instrumentation monitors process variables, such as pressure, temperature, level, and flow in a manner typical of chemical processes. The main difference in process control instrumentation for pharmaceutical processes is in the interface between the sensing element or device and the clean process stream. Analytical chemistry instrumentation monitors the chemistry of the process throughout the manufacturing process. It is especially important during the purification, compounding, and formulating of the finished product. Sample collection and conditioning—prior to introduction into the analytical instrument—is critical to reliable analysis, in both laboratory devices and in at-line and on-line analyzer systems. The cleanliness of the sampling system is an important element of reliability and repeatability. Cleanliness is impacted by the design, manufacture, and condition of the fluid control components used in the system. Consideration must be given to potential inboard, outboard, and internal leakage; effective sealing systems; entrapment; material selection; surface finishing and conditioning; and cleanability. The result of these considerations is an evaluation process that will lead to selection of the analytical chemistry instrumentation system components that will optimize repeatability and make validation easier. Component Selection Process Proper selection of components for service in clean systems is the responsibility of the system designer. Following are some elements for consideration in the selection process: Design Choose components that have proven leak-tight performance capabilities, considering potential for inboard, outboard, and internal leakage. Consider the seals used in the design. Select components with sealing systems that will meet the system’s service conditions. Look at the potential for entrapment and select components designed to minimize entrapment areas. Study the flow path through the component. Consider the component with the smoothest flow path and minimal internal volume. Consider the materials of construction and of the seal members. Be certain that they will be compatible with the pressure, temperature, and media requirements of the system. Check all ratings for compatibility with system requirements. Manufacture Check surface finishes. Components with the best surface finishes will be easier to clean and keep clean. Consider how the components are assembled and the possibilities for entrapment. Determine if the lubricants used will be compatible with the system. Cleaning Study the cleaning method used by the manufacturer and select components that will meet the system requirements initially and in service. Verification Determine what certifications will be required to verify the necessary elements of the specification or procurement document. Packaging Specify the packaging method that best meets the need of the end user.

Updated guidance on requirements for paediatric 'compliance checks' during validation

We have previously published guidance on how the MHRA will handle the impact of the requirements of the Paediatric Regulation on the validation of applications for marketing authorisations (MAs). The guidance has been updated to reflect the need for compliance checks on certain line extensions and variations as well as initial applications for new medicinal products.
The same principles apply in these cases and the MHRA will continue to request an opinion from the European Paediatric Committee where one has not already been obtained by the applicant. The document has also been updated to refer to the final European Commission guideline on the content of applications for paediatric investigation plans and the operation of the compliance check and also to other European Medicines Agency (EMA) and Co-ordination Group for Mutual Recognition and Decentralised Procedures (CMDh) guidance.
Guidance on the MHRA’s Handling Of The Requirement in the Paediatric Regulation to Undertake a ‘Compliance Check’ During Validation (85Kb)

Is FDA's Draft Process-Validation Guidance a Mixed Blessing?

By: Erik Greb

The US Food and Drug Administration’s Draft Guidance for Industry—Process Validation: General Principles and Practices provides a life-cycle approach for validating pharmaceutical processes and aims to help pharmaceutical companies achieve consistently high product quality. The document includes several concepts that are familiar to the industry but also contains ambiguities and recommendations that might be difficult for some drugmakers to follow.
The draft guidance suggests manufacturers establish links from their clinical process to their commercial-manufacturing process. This approach is similar to the one FDA has used in its preapproval inspections. If the guidance becomes final as it currently stands, manufacturers may be expected to use the data that they gain during formulation and development to define a product’s critical attributes, which would be the basis for the manufacturing-process parameters.
The agency points out that development and formulation data can improve a company’s understanding of its processes during scale-up and commercial manufacturing. This understanding would help companies control variability and increase product quality, says Chris Ames, director of global validation at Catalent Pharma Solutions (Somerset, NJ). Companies would submit these data to FDA to establish links between clinical and commercial processes.
But the draft guidance does not advise manufacturers about how to identify the most important characteristics of its product or manufacturing process, or about how to demonstrate links from the clinical to commercial process. “They’ve left it completely open to interpretation as to what data you provide and what format you use,” says Jim Agalloco, president of Agalloco and Associates. This ambiguity would suit Big Pharma because it frees companies to use their experience and discretion in deciding how to follow the guidance, says Agalloco. Small and emerging drugmakers, however, would likely be confused because they don’t have the depth of knowledge that would help them define critical attributes.
Some elements of the draft guidance resemble a Six Sigma approach to manufacturing, which is familiar to the pharmaceutical industry. The main similarity is the draft guidance’s recommendation of a statistical link that demonstrates that variability remains constant from the clinical through the commercial manufacturing stages. The statistical link is intended to confirm that processes are the same throughout all phases.
Although the draft guidance suggests statistical analysis, it leaves industry with only a broad understanding of what that means. FDA does not explicitly suggest that manufacturers use particular statistical tools, the agency simply recommends that companies apply good statistics to establish the links, says Agalloco.
The draft guidance suggests manufacturers define a process that can be measured, analyzed, improved, and controlled, and this approach is closely related to Six Sigma. The benefit of the Six Sigma technique is that it provides a mechanism for scientific review of a process, for assessing variability, and for identifying improvements, says Ames.
On the other hand, it is unclear whether the draft guidance recommends a product be refined in the way that a Six Sigma approach would. “To me, Six Sigma implies an acceptance by FDA that you might not have done a sufficient job in development and scale-up and are allowed to improve product and process while it is in operation,” says Agalloco. Patients’ experiences with a product might persuade a manufacturer that it should adjust one of the drug’s parameters to improve it. Six Sigma would allow postcommercialization changes to a product, but the draft guidance may not be compatible with them, Agalloco says.
Before it could submit a regulatory filing, a company would have to spend a great deal of time and money to better understand its ingredients, its product, its manufacturing process, its material handling, and associated variables. Pharmaceutical companies might object to the draft guidance’s approach because it suggests this expensive work be completed before commercialization, but the costs would not be recoverable before commercial-scale manufacturing began.
Although it is based on good science, if the final guidance is approved as drafted, it could easily increase drug-development time by one or two years, thus costing a manufacturer millions of dollars, says Warren Charlton, a consultant at WHC Bio Pharma Technical Services. Manufacturers would need to use smart strategies to shorten development time, but not knowing how much data regulators expect in a submission would make this strategy difficult.
The draft guidance inspired a huge volume of comments that will likely take FDA a long time to review, says Agalloco. Even though the guidance might not be final for at least a year, manufacturers would be wise to study it now and seek advice about interpreting it. In this difficult time for the pharmaceutical industry, no company can afford to ignore regulators’ recommendations, and advance preparation would be to a manufacturer’s benefit.

Contractee Responsibilities in Outsourced Pharmaceutical Quality Control Testing

aMethod Qualification (Verification and/or Validation)Once a contract testing laboratory has been given “approved” status, the actual methods to be used may be qualified for their purpose. If the contractor has a desired compendial method in place, the contractee is responsible for providing a sample of test material for verification according to compendial requirements. The US Pharmacopeia includes guidance on verification of compendial procedures (16). For noncompendial lot-release methods (e.g., viral and mycoplasma testing, which are driven by US FDA points to consider and ICH guidance documents), the methods will require validation if used for GMP purposes.
Validation is normally performed by a contract testing organization with a generic sample matrix, so it should not be confused with the product-specific qualification described below. Validation of compendial procedures is outlined in the US Pharmacopeia (17) as well as the International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) (18). A contractee may wish to confirm the status of a given method by evaluating validation reports during an on-site visit. Contract testing labs may also be willing to provide copies of method validation reports or summary documents describing those results.
Commercial product lot release testing is expected to be performed using methods deemed suitable for a given product. This typically entails product qualification for the analytical method and evaluation of possible matrix interference. The contractee is responsible for commissioning such studies before routine use of any method and should maintain the resulting reports as evidence that the methods being used are suitable for their purpose. Such studies may need to be repeated if the processes involved in manufacturing a commercial product are modified significantly. Quality and Business Agreements Regulators expect that the relationship between a product sponsor and its contract testing partner be formalized by a quality agreement (7,8). Such agreements specify explicitly the responsibilities of each partner and provide the means by which a contractee extends its QC testing standards to a contractor. The agreement should list technical and/or quality contacts (names and phone numbers) at the contract testing organization as well as contact information for appropriate decision makers at the contractee organization.
Among the specifics to be detailed in a quality agreement are

• the types of compliance to be followed in contracted studies
• details on interactions between the contractor's and the contractee's quality systems
• requirements for equipment and assay validation, verification, and/or qualification
• assurance of quality and reporting requirements
• data recording and archiving practices
• conduct of investigations into nonconformances, deviations, unexpected, and OOS results (and timing of client notification)
• notification of regulatory inspection
• requirements for use of subcontracting laboratories
• use of debarred personnel
• availability of the contract testing laboratory for periodic technical and compliance as well as for-cause audits.

Business requirements should be addressed in a separate document and may take the form of a master service agreement, an agreement of scope, a pricing agreement, or a standard terms and conditions agreement supplied by the contractor. Such considerations may include assay pricing (including discount structures), assay initiation and report turn-around times, testing volume and exclusivity, availability of rush service and associated charges, and (in some cases) penalties for late reporting. These business agreements often specify the requirements of a contract testing laboratory with respect to contractee notification of impending sample submission, which are detailed below.