Downstream processing validation

Most of these methods are commonly employed in the downstream processing of the desired ceU culture technology product. Hence, most of the time it is only necessary to demonstrate that the designed process is reducing the putative risk factors to acceptable levels. Validation methods employed for risk reduction are discussed in the Hterature (25). 

Several key issues have to be addressed in the downstream processing of biopharmaceuticals regardless of the expression system. The removal of host cell proteins and nucleic acids, as well as other product- or process-related or adventitious contaminants, is laid down in the regulations and will not differ between the individual expression hosts. The identity, activity and stability of the end product has to be demonstrated regardless of the production system. The need for pharmaceutical quality assurance, validation of processes, analytical methods and cleaning procedures are essentially the same. 

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. 

For more comprehensive validation studies, the molecular mass profile of the DNA spike should roughly approximate to the molecular mass range of endogenous contaminant DNA in the crude product. Obviously, the true DNA clearance rate attained by downstream processing procedures (e.g. gel filtration) will depend to some extent on the molecular mass characteristics of the contaminant DNA. 

Figure 6.3 Nonspecific protein adsorption as a result of column degradation. White circles indicate eluted column peak heights of a new column. Black diamonds indicate eluted peak heights after the column was treated with 50 wash cycles. See text for discussion. (Data from P. Gagnon, 1997, Validated Biosystems Quarterly Resource Guide for Downstream Processing, 2(1), 1, http //www.validated.com/revalbio/library.html.)...

P. Gagnon, 1997, Validated Biosystems Quarterly Resource Guide for Downstream Processing, 2(1), 1, http //www.validated.com/revalbio/library.html. 

Reif, O. W., Solkner, P., Rupp, J. Analysis and evaluation of filter cartridge extractables for validation in pharmaceutical downstream processing. J Pharm Sci Tech 50 399-410 (1996). 

The subsequent downstream processing section, which includes visualization of quantified proteins, statistical validation of differences in treatments or samples, and biological interpretation, is much less defined in terms of work-flow regimens and is discussed toward the end of this chapter. In the succeeding text, various relevant aspects of proteomic workflows that impinge on the data obtained from proteomic analysis of prokaryotes assuming that a gel-free approach is used are discussed. 

Process validation starts with the identification of product quality attributes and justification of acceptance criteria, followed by a review of the risk analysis, execution of process development runs, and compilation of clinical material manufacturing data to set specifications considering process variability [11]. There is a greater focus on process validation for downstream steps rather than for upstream steps because downstream steps are associated with virus removal. Process validation is just one approach used to control virus contamination, however others include cell bank characterization, in-process testing, inactivation procedures, control of raw materials, containment, and postmarket surveillance [6]. 

Fermentation. A validatable fermentation process demonstrates a controllable method of growing cells that reliably express the biopharmaceutical product that can be reproducibly recovered if broth is harvested within a set of specified conditions that allow the product to meet its quality specifications [10]. The goals of fermentation process validation are to provide documented evidence that all aspects of the process perform as intended to generate consistent fermentation broths at harvest this consistency in turn permits downstream steps to yield a... 

To link fermentation process input parameters to output parameters, specifically behavior in downstream isolation steps and ultimate product quality, usually a partial or total purification is necessary, which requires substantial labor to process samples [10], Sample evaluation also is limited depending on the level of product characterization. Consequently, fermentation process validation has been viewed as less rigorous compared with other forms of process validation [10],... 

At the preclinical product phase, critical and noncritical classification of process input parameters should be initiated [32]. Critical components of facility subsystem validation need to be essentially complete before phase I product manufacture [15]. For phase I, it is necessary to validate aspects of the process related to product safety (e.g., sterility, mycoplasma, viral clearance, impurity removal, and stability) [14]. Abbreviated viral clearance studies for model viruses/retroviruses and impurity clearance studies for host cell DNA often are acceptable, resulting in fewer downstream steps validated at this product stage [3, 5]. If viral clearance results are available in sufficient time, the results can be applied to developing the phase I process steps. All assays do not have to be validated at this stage, but some (especially product-specific ones) should be at least qualified [14]. 

Walter J, Allgaier H (1997). Validation of downstream processes. In R Wagner, H Hauserv (eds.), Mammalian Cell Biotechnology in Protein Production Walter De Gruyter Inc., New York, pp. 453-482. 

One cornerstone provision of cGMPs is validation, a concept introduced to assure product consistency [84], The validation of downstream processing operations aims to prove that they are capable of consistently removing impurities (e.g., host cell components, process-related materials, adventitious agents) to acceptable levels. Additionally, acceptance limits and operating ranges for each step must be determined [84]. Validation studies usually lead to optimized processes with reduced variability and, as a consequence, to a decrease in the number of failed batches. Thus, the development of adenovirus manufacturing processes (and associated facility) should be undertaken with validation in mind, not only to improve quality assurance and accelerate approval, but also to reduce costs [85, 86]. 

DNA Validation and Quantification It is important to know how successful the DNA extraction process was. The quality, quantity, and length(s) of DNA should be estimated prior to downstream processing. Here, we describe validation and quality measurement using a Nanodrop machine. 

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