Biopharmaceuticals protocols

The physicochemical and other properties of any newly identified drug must be extensively characterized prior to its entry into clinical trials. As the vast bulk of biopharmaceuticals are proteins, a summary overview of the approach taken to initial characterization of these biomolecules is presented. A prerequisite to such characterization is initial purification of the protein. Purification to homogeneity usually requires a combination of three or more high-resolution chromatographic steps (Chapter 6). The purification protocol is designed carefully, as it usually forms the basis of subsequent pilot- and process-scale purification systems. The purified product is then subjected to a battery of tests that aim to characterize it fully. Moreover, once these characteristics have been defined, they form the basis of many of the QC identity tests routinely performed on the product during its subsequent commercial manufacture. As these identity tests are discussed in detail in Chapter 7, only an abbreviated overview is presented here, in the form of Figure 4.5. 

Figure 4.9 Scale-up of proposed biopharmaceutical production process to generate clinical trial material, and eventually commercial product. No substantive changes should be introduced to the production protocol during scale-up...

An investigational new drug is a new chemical-based, biologic or biopharmaceutical substance for which the FDA has given approval to undergo clinical trials. An IND application should contain information detailing preclinical findings, method of product manufacture and proposed protocol for initial clinical trials (Table 4.9). 

Contaminant-clearance validation studies are of special signibcance in biopharmaceutical manufacture. As discussed in Section 7.6.4, downstream processing must be capable of removing contaminants such as viruses, DNA and endotoxin from the product steam. Contaminant-clearance validation studies normally entail spiking the raw material (from which the product is to be purihed) with a known level of the chosen contaminant and subjecting the contaminated material to the complete downstream processing protocol. This allows determination of the level of clearance of the contaminant achieved after each purihcation step, and the contaminant reduction factor for the overall process. 

Methods development starts with a relatively high number of techniques to characterize and test samples. The number of protocols is often reduced once the critical parameters and the methods that identify them have been defined. The analyst must evaluate the initial techniques with respect to their purposes. If the goal is to generate research data, the practicality of the method and its limitations are not of primary concern if the goal is to use the technique as part of a test procedure, it has to be evaluated in terms of its potential to meet full validation. Critical procedures (e.g., release testing) that cannot be validated will bring a project to an expensive halt. For these reasons, this chapter provides basic principles as well as limitations of capillary electrophoresis (CE) as applied to the analysis of real biopharmaceutical molecules. 

Both pre-clinical and clinical trials are underpinned by a necessity to produce sufficient quantities of the prospective drug for its evaluation. Depending on the biopharmaceutical product, this could require from several hundred grams to over a kilo of active ingredient. Typical production protocols for biopharmaceutical products are outlined in detail in Chapter 3. It is important that a suitable production process be designed prior to commencement of pre-clinical trials, that the process be amenable to scale-up and, as far as is practicable, that it is optimized (Figure 2.9). The material used for pre-clinical and clinical trials should be produced using the same process by which it is intended to undertake final commercial-scale manufacture. 

The cleaning of process-scale chromatography systems used in the purification of biopharmaceuticals can also present challenges. Although such systems are disassembled periodically, this is not routinely undertaken after each production run. CIP protocols must thus be applied periodically to such systems. The level and frequency of CIP undertaken will depend largely on the level and type of contaminants present in the product-stream applied. Columns used during the earlier stages of purification may require more frequent attention than systems used as a final clean-up step of a nearly pure protein product. While each column is flushed with bulfer after each production run, a full-scale CIP procedure may be required only after every 3-10 column runs. Most of the contaminants present in such columns are acquired from these previous production runs. 

Biopharmaceuticals have completely different properties from conventional low molar mass pharmaceuticals. This particularly affects product characterization, due to the complex production processes and protein structures. Thus, successful production of biopharmaceuticals relies mainly on strict protocols, clinical expertise, and follow-up during clinical application (Crommelin et al., 2003). 

ADDING PREDICTIVE SAFETY BIOMARKERS INTO STANDARD TOXICOLOGY PROTOCOLS FOR BIOPHARMACEUTICALS... 

The usual GLP 30- or 60-day repeat-dose toxicology study with a recovery group offers an opportunity to perform a more systematic investigation of the more subtle pharmacodynamic or toxicologic effects of biopharmaceuticals than those endpoints usually incorporated into such protocols. Some of these demand tissue samples, but many involve noninvasive biomarkers that can be carried forward into early phase human studies. These might include CNS assessments, inflammation and immune activation or suppression, cell proliferation or apoptosis in tissue samples, and end-organ toxicities. 

The safety pharmacology studies in which the respiratory, CNS, and cardiovascular systems are evaluated in a nonrodent model are not de facto for a biopharmaceutical. Quite frequently the safety endpoints are in the protocols for the pivotal repeat-dose studies, such as a cynomolgus study, obviating the need for separate safety pharmacology studies. 

Included in the first portion of this section should be an overall tabulated summary of all in vivo biopharmaceutical studies carried out on the drug grouped by type of study. The study number, route of administration, dosage form, batch number, plant and date of manufacture, number of subjects, IND or NDA number under which the study was conducted or study submitted, date of submission, conclusions regarding the study, and previous agency response on the study or the protocol together with the date of the correspondence should be included. 

When bringing the utilities to the point of use, care should be taken to ensure that the clean room is not compromised. A clean construction protocol should be implemented and wall, ceiling, and floor penetrations, if needed, should be flashed and sealed in such a manner as to prevent contaminants from entering the clean room. Such entry points should also be smoothly sealed to ensure that there are no crevices to harbor organisms. Drains should be avoided in the clean room wherever possible. When this is not possible, the drains should be covered when not in use with a means specifically designed for biopharmaceutical clean-room application. Such means are tight, smooth, cleanable, and corrosion resistant. 

Ongoing experience has demonstrated that an aggressive clean construction protocol program is generally not required for biopharmaceutical facilities that do not... 

The ability efficiently to manipulate DNA elements afforded by the Gateway cloning system has opened (see also Part III, Chapters 1 and 7) - and wiU continue to open - many doors to novel experimental protocols that wiU be invaluable in basic research finked with the development of modem biopharmaceuticals. 

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