April 1, 2006 (Vol. 26, No. 7)

Globally Harmonized Regulatory Approach Is Needed to Move Forward Expeditiously

The concept of well-characterized biologicals originated with the FDA Center for Biologics Evaluation and Research (CBER), which first employed the term in 1996. Prior to 1996, CBER required both a Product License Agreement (PLA) and an Establishment License Agreement (ELA) for product approval. The ELA was specific to the facility and process used for that product. It was written to prevent unapproved process variations.

For complex biological products, such as blood components, the constraints of the PLA/ELA combination helped ensure safe and reproducible manufacturing standards. As the technology for characterization of molecular diversity has evolved, the concept has been further refined. For protein pharmaceuticals, well-characterized meant that the natural molecular heterogeneity, impurity profile, and potency could be defined with a high degree of confidence.

The Biological License Application (BLA) places primary emphasis on the detailed analytical description of the molecular entity (well-characterized) as the vehicle for systematization of the reproducible manufacture of the biological pharmaceutical. The term well-characterized biological has now been abandoned in favor of the more precise specified biological.

Plasmid DNA

&#8220When producing a biophysically well-defined entity, the process does not have to be considered the product,&#8220 states Sangeetha Sagar, Ph.D., senior director of the biologics development and engineering, bioprocess R&D group at Merck (www.merck.com). &#8220For example, vaccines and gene therapy products using plasmid DNA can be well-characterized in terms of structure, expression, and purity.&#8220

Consequently, when classic methods of manufacture become scale limiting or prohibitively expensive, novel process technologies can be employed as long as final product quality is maintained. For early research purposes, traditional ultracentrifugation-based purification methods are sufficient to meet the milligram quantity requirement. For formulation development, product safety studies, and boutique commercial product needs, a chromatography-based process can deliver in the 10&#8211100-g range.

Dr. Sagar stresses the need for creative nonchromatographic processes for high-dose vaccine products for worldwide markets to overcome cost and scale limitations. The Merck group has developed an inexpensive precipitation technique, involving incremental use of selective precipitating agents. This strategy resulted in highly purified plasmid product without resorting to anion exchange or other chromatographic methods. Robust separation of the plasmid DNA from host contaminants was achieved.

With proper release and characterization methods in place, Dr. Sagar proposes that unique manufacturing processes can be used, based on the scale and commercial needs of the product with changes made in the course of product development.

Conjugate Vaccines

Thorough characterization of polysaccharides is critical for effective conjugate vaccine development to monitor identity, purity, and potency, according to John Kim, Ph.D., (www.wyeth.com) vaccine R&D. The firm has a program aimed at Streptococcus pneumoniae. The organism contains a highly immunogenic coat, consisting of different families of polysaccharides. Conjugated pneumococcal vaccines are based on mixtures of the polysaccharides covalently attached to carrier proteins.

Wyeth&#8217s Prevenar contains seven serotypes, individually conjugated to a carrier protein. Newer vaccines are under development with additional serotype coverage requiring highly sophisticated instrumentation, including high-performance anion exchange chromatography with pulsed amperometric detection, reverse-phase HPLC with fluorescent detection, and nuclear magnetic resonance.

To enlarge the scope of their characterization protocols, the Wyeth group has applied gas chromatography mass spectrometry (GC-MS) to the detailed characterization of the 13-valent pneumococcal polysaccharides and polysaccharide-protein conjugates.

The results obtained were comparable to current, validated/qualified colorimetric assays. However, the coverage of the samples is broader, as GC-MS could be applied for analysis of early- and late-stage samples. The GC-MS methodology revealed critical properties, including the location and order of activation sites, monosaccharide composition and linkage profiles in the final drug product. This is due to the excellent resolution of the monosaccharide derivatives combined with extremely specific low-level detection.

Dr. Kim and his colleagues have determined that the GC-MS provides superior specific information concerning peak purity and spectral analysis to that obtained with other technologies. These data are critical to define these vaccines as well-characterized biologicals.

Mabs from a Scale-Up Process

Machinani J. Rao, Ph.D., associate director in analytical development at PDL BioPharma (www.pdlbiopharma.com), stresses that an extensive data set is important to establish the historical comparability of the drug substance in the event of process or facility changes.

During the scale-up process the compound under investigation may end up with a new impurity profile or with altered quality and efficacy attributes. According to Dr. Rao, PDL recently upgraded the manufacturing process for a humanized Mab and transferred this process to a new production site.

A detailed physicochemical, conformational, and biological characterization of a commissioning lot was produced from the scale-up process-development campaign. The characterization included a battery of tests to define the primary, secondary, and tertiary structure of the antibody. The structural integrity of the commissioning lot was also assessed using a reference standard lot produced from the earlier scale production process.

The Mab glycosylation profile, monosaccharide composition, and post-translation modifications, including relative charge variant levels were also measured in this study. The extent of light-chain clip formation, aggregate, and deamidation levels were similar to levels observed for earlier lots from the current process scale. A slight increase in N-terminal variant level was observed in the commissioning lot produced using the new process.

One Set of Specifications?

&#8220A globally harmonized regulatory approach for specifications of well-characterized biological products would be desirable for both pharmaceutical manufacturers and regulatory authorities,&#8220 states Laura Bass, Ph.D., of Pfizer (www.pfizer.com). Currently, for a given product marketed worldwide there may be differences in requirements for tests, analytical procedures, and/or acceptance criteria between regulatory regions. This makes development of a global set of specifications an extremely challenging task.

Whereas the ideal scenario, according to Amy Dingley of Stryker Biotech (www.stryker.com), would be a single international specification for raw materials and intermediates, process control, release, and expiry, the real-world situation is more challenging.

Differing compendial requirements for raw materials, differing requirements for in-process and release specifications, and a staggered approach to registration can result in a plethora of registration rules for a single product. Stryker&#8217s strategy is to maximize the product similarities at the upstream end, pushing the differences further downstream to gain flexibility.

The process of setting specifications for a biologic suffers from varying interpretations by regulatory bodies, making global harmonization a distant goal rather than an immediate possibility, states Wassim Nashabeh, Ph.D., of the department of analytical chemistry at Genentech (www.genentech.com).

Dr. Nashabeh proposes an integrated lifecycle approach through all phases of product development. Key features of the lifecycle strategy include the characterization of clinical and nonclinical attributes specifying the clinical relevance and clinical ranges. In addition, the process capability and manufacturing consistency need to be taken into account.

The characterization of biologics is progressing rapidly, thanks to advances in instrumentation and the expanding ability of scientists to detect finer levels of divergence among products. However, advances in the laboratory will have to be matched by standardization of international regulatory guidelines if the worldwide sharing and marketing of biologicals is to move forward expeditiously.

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