Introduction

Downstream processing serves to (a) recover the therapeutic protein from its producer cell source upon completion of the upstream processing phase, (b) purify the protein and (c) formulate the protein into final product format.

An overview of the steps normally undertaken during downstream processing is presented in Figure 6.1. Details of the exact steps undertaken during the downstream processing of any specific biopharmaceutical product are usually considered confidential by the manufacturer. Such details are thus rarely made generally available. However, a potential downstream processing procedure for recombinant tPA is presented in Figure 6.2, and other examples are provided at various stages through the remainder of this text.

Downstream processing is undertaken under clean-room conditions in order to protect the product stream from environmental contamination (Figure 6.3). In addition, the water used as solvent during downstream processing (and, indeed, often during upstream processing) is highly purified 'water for injections' (WFI). Standard potable (drinkable) water contains contaminants (e.g. microorganisms, dissolved organic and particulate matter, etc.) that could either react with the protein directly or that would have an adverse effect upon patient health if present in the final product. Generation of 'purified water' (often used to make up media for microbial fermentation) along with even purer WFI, is summarized in Figure 6.4.

All proteins retain their structural integrity and biological activity only over characteristic pH ranges. Proteins become denatured outside these ranges, losing their characteristic three-dimensional structure, and hence activity (Chapter 2). Most biopharmaceuticals are stable only at pH values approaching neutrality (approximately pH 5-8 for many). As such, downstream processing is carried out using not WFI per se as solvent but in buffer solutions made from WFI. A buffer is a solution that resists a change in its pH value even with the addition of small amounts of either acid or alkali, and hence effectively controls the pH environment of the protein.

Pharmaceutical biotechnology: concepts and applications Gary Walsh © 2007 John Wiley & Sons, Ltd ISBN 978 0 470 01244 4 (HB) 978 0 470 01245 1 (PB)

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

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

Figure 6.2 A likely purification procedure for tPA produced in recombinant E. coli cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltration step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed, as tPA is known to bind immobilized lysine molecules. Adapted with permission from Bio/Technology (1993), 11, 351

Figure 6.2 A likely purification procedure for tPA produced in recombinant E. coli cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltration step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed, as tPA is known to bind immobilized lysine molecules. Adapted with permission from Bio/Technology (1993), 11, 351

Figure 6.3 Photograph illustrating a typical pharmaceutical deanroom and some of the equipment usually therein. Note the presence of a curtain of (transparent) heavy-gauge polyethylene strips (most noticeable directly in front of the operator). These strips box off a grade A laminar flow work station. Product filling into final product containers is undertaken within the grade A zone. The filling process is highly automated, requiring no direct contact between the operator and the product. This minimizes the chances of accidental product contamination by production personnel. Photograph courtesy of SmithKline Beecham Biological Services, s.a., Belgium

Figure 6.3 Photograph illustrating a typical pharmaceutical deanroom and some of the equipment usually therein. Note the presence of a curtain of (transparent) heavy-gauge polyethylene strips (most noticeable directly in front of the operator). These strips box off a grade A laminar flow work station. Product filling into final product containers is undertaken within the grade A zone. The filling process is highly automated, requiring no direct contact between the operator and the product. This minimizes the chances of accidental product contamination by production personnel. Photograph courtesy of SmithKline Beecham Biological Services, s.a., Belgium

Incoming potable water

Purified water

Figure 6.4 Overview of a generalized procedure by which purified water and WFI are generated in a pharmaceutical facility. Refer to text for specific details

Purified water

Figure 6.4 Overview of a generalized procedure by which purified water and WFI are generated in a pharmaceutical facility. Refer to text for specific details

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