Downstream processing, biopharmaceuticals

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. 

This chapter aims to overview the manufacturing process of therapeutic proteins. It concerns itself with two major themes (1) sources of biopharmaceuticals and (2) upstream processing. The additional elements of biopharmaceutical manufacturing, i.e. downstream processing and product analysis, are discussed in Chapters 6 and 7 respectively. 

Overall, therefore, the routine manufacture of a biopharmaceutical product is initiated by large-scale culture of its producing cell line (upstream processing). Subsequent to this, the product is recovered, purified and formulated into final product format. These latter operations are collectively termed downstream processing and are described in Chapter 6. 

Figure 6.1 Overview of a generalized downstream processing procedure employed to produce a finished-product (protein) biopharmaceutical. QC also plays a prominent role in downstream processing. Qualty control personnel collect product samples during/after each stage of processing. These samples are analysed to ensure that various in-process specifications are met. In this way, the production process is tightly controlled at each stage...

Biopharmaceutical products are also subjected to screening for the presence of viral particles prior to final product release. Although viruses could be introduced, for example, via infected personnel during downstream processing, proper implementation of GMP minimizes such risk. Any viral particles found in the finished product are most likely derived from raw material sources. Examples could include HIV or hepatitis viruses present in blood used in the manufacture of blood products. Such raw materials must be screened before processing for the presence of likely viral contaminants. 

Incorporation of downstream processing steps known to inactivate a wide variety of viral types provides further assurance that the final product is unlikely to harbour active virus. Heating and irradiation are amongst the two most popular such approaches. Heating the product to between 40 and 60°C for several hours inactivates a broad range of viruses. Many biopharmaceuticals can be heated to such temperatures without being denatured themselves. Such an approach has been used extensively to inactivate blood-borne viruses in blood products. Exposure of product to controlled levels of UV radiation can also be quite effective, while having no adverse effect on the product itself. 

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. 

The ELISA is a versatile method that can be used throughout the biopharmaceutical product development process, from small-scale research to cell-line selection, to monitoring fermentation and downstream processing, and to product release testing. The use of the microtiter plates allows for high sample throughput and various degrees of automation. ELISAs satisfy the biopharmaceutical production requirements for specific, accurate, precise, and reproducible assays. 

The ELISA can be used for identification and quantitation of the protein product (biopharmaceutical) of interest throughout the development, production, and manufacturing process. For example, in the initial development phase, ELISAs can aid in the selection of the best cell line. In the early manufacturing steps, it can be used to identify the appropriate product-containing pools or fractions in process to be subjected to further purification. Because of the selectivity of ELISA, it is a suitable tool to select out the protein of interest from complex protein mixtures, such as cell culture fermentation media or product pools in early steps of protein recovery as well as downstream processing. Even complex mixtures do not require much sample preparation. It is important to determine... 

An overview of the steps normally undertaken during downstream processing is presented in Figure 3.16. Details of the exact steps undertaken during the downstream processing of any specific biopharmaceutical product are usually considered highly confidential by the... 

The extraction and purification of proteins from organisms or biological tissue can be a laborious and expensive process, and often represents the principal reason why vaccines and other therapeutic agents reach costs that become unattainable for many. Downstream processing also can be a major obstacle with respect to cost for large-scale protein manufacturing in plants. However, purification from plant tissues, while still costly, is in general less expensive than purification from their mammalian and bacterial counterparts. Indeed, some plant-derived biopharmaceuticals, such as topically applied monoclonal antibodies, may require only partial purification and thus be even less intensive in terms of labor and cost. 

Downstream processing involves employment of a purifying system that can isolate the product in as few steps as possible using the simplest purification technology that will achieve the required purity. While purity is a critical consideration for both small-molecule pharmaceuticals and biopharmaceuticals, the nature of biopharmaceutical administration (typically via injection) and the nature of biotechnology processes require that additional considerations be paid to the purity of biopharmaceuticals. The final product must meet regulatory purity and sterility standards and must be below the maximally acceptable cellular or microbial contamination (Ho and Gibaldi, 2003). 

TFF is widely applied in biopharmaceutical downstream processes including concentration, clarification, fractionation, and diahltration. 

Downstream processing for virtually aU protein biopharmaceuticals follows a fairly predictable sequence of events (outlined in Fig. 1) [17]. Following initial product recovery and concentration, multiple chromatographic steps are undertaken (usually between three and six individual fractionation steps). While gel filtration and ion exchange are particularly common, down-... 

---