Overview Of The Aims Of Purification

The use of biological medicines in humans requires that they are purified to standards determined by regulatory authorities primarily to ensure patient safety, but also to ensure that product is consistent and effective. The standards relating to purification are used to determine the permissible levels of impurities in the administered product, and hence the specifications that are set for the product during development and manufacture (Seamon 1998). In beginning to devise a purification strategy for any biological medicine, it is vital to know the proposed dose, dose schedule, product concentration and route of administration, in order to interpret the regulatory guidelines and set limits for the expected impurities such as DNA, host cell protein and endotoxin.

Impurities, such as endotoxin or DNA, are usually limited to a certain specified amount per dose, so the product purity will be different depending on the amount of product in the dose. For example, a dose of erythropoietin may be only 10 |ig, whereas the dose for albumin could be as high as 50 g. In either case the allowable level of endotoxin or DNA, per dose, would be the same. The purity per unit mass of product will be much higher where a greater amount of product is administered in a single dose.

It is important that assays for quality control testing of product impurities and process contaminants are available throughout purification development. A useful distinction can be made between impurities that exist in the process start material (fermentation harvest) such as host cell protein or DNA, and contaminants derived from processing materials that are introduced during the purification process such as leached protein A, or Tween used, perhaps, to prevent aggregation. Table 18.1 gives examples of quality control tests that may be employed at various stages in the downstream process of a product derived from animal cells.

Assays for product integrity and activity are also essential during process development. It is vital to know that the conditions and operations used to purify the product do not cause instability or inactivation. Temperature and pH may be critical to product breakdown or modification, and may cause the product to lose activity. Equally, high concentrations of product, either in solution or when loaded onto a chromatography column, could cause product aggregation. The presence of proteases from the cell culture may cause product degradation and loss of activity. It is important to have assays that can distinguish active, non-degraded product, and that these are used to determine the integrity of the product during the process. It will be important to define the conditions, for example temperature and pH ranges, within which product remains stable and for how long exposure to adverse conditions is acceptable.

Medicines from Animal Cell Culture Edited by G. Stacey and J. Davis © 2007 John Wiley & Sons, Ltd

Table 18.1 Examples of Quality Control testing employed at various stages in the downstream process.

Process Stage

Description

Examples of QC Testing

Raw Materials

Column matrices, membranes,

Appearance, chemical identity, endotoxin

chemical reagents, buffers,

content, package integrity, correct

excipients

certification

Crude Harvest

Unpurified product from cell

Product concentration (e.g. by ELISA), total

culture

protein concentration (e.g. by Lowry or Bradford), SDS-PAGE, pH, conductivity, adventitious agents, mycoplasma, Reverse Transcriptase

Product Intermediates

Column eluates, eluate fractions,

Product concentration (e.g. by ELISA),

filtrates, concentrates

total protein concentration (e.g. by Lowry, Bradford or OD280), SDS-PAGE, measurement of specific impurities for pooling of fractions, pH, conductivity

Drug Substance

Purified bulk product

Appearance, pH, HPLC (e.g. size exclusion or reverse phase), DNA, SDS-PAGE, Western blot, isoelectric focussing, N-terminal sequencing, endotoxin, bioburden, trace metals, potency

Drug Product

Formulated, filled product (e.g.

Sterility, pH, appearance, endotoxin,

in vials, ampoules or pre-

volume in container, strength (product

filled syringes)

concentration), osmolarity, potency

Purification processes are normally built from steps that can be categorized as follows (in sequential order of their use):

• clarification - the removal of cells and cell debris at the start of the process;

• capture - the removal of gross contamination and concentration of product, usually by chro-matography;

• intermediate step - the conditioning of the product to prepare for the next process step, for example buffer exchange by diafiltration to remove salt prior to loading onto ion exchange, or to concentrate product prior to size exclusion chromatography;

• purification - removal of the bulk of the impurities, usually by chromatography;

• polishing - removal of trace impurities and contaminants, yielding drug substance (or bulk purified product);

• formulation and fill - putting the product into the right buffer and filling into the final containers for patient administration, yielding drug product.

These steps can be built into a coherent process, where each step has a purpose, or purposes. An example of such a process flow is given in Figure 18.1.

It is often difficult to categorize a purification step. For example, Protein A is often used as a capture step in antibody purification, but, as an affinity step, it also removes the bulk of the impurities. However the steps are categorized, each step should always have a defined purpose in the purification scheme, and should be justified by data supporting the need for, and effectiveness of, the step in the process.

OVERVIEW OF THE AIMS OF PURIFICATION 349

Crude Harvest

Crude Harvest

Example Purification Flow Chart
Drug Product Figure 18.1 Example Process Flow Diagram.

Practical guides in the use of purification methods for biological products can be useful; some are intended for laboratory-scale work (Harris & Angal 1995a,b) and give guidance in the use of, for example, chromatographic techniques; others are aimed at large-scale bioprocessing (Subra-manian 1998). Useful guides can be obtained from the suppliers of membranes and chromatography matrices, such as the series by GE Healthcare or The Busy Researcher's Guide to Biomolecule Chromatography by Applied Biosystems.

It is also necessary to know the scale at which product will need to be made for commercial supply, as this will influence the way the process has to be developed. The number of doses per year and the amount of product per dose should be known at the outset, and will be a key part of the business plan. A large volume product will generally need to be produced more cheaply, on a cost per gram basis, and the cost of goods will be vital to the commercial viability. A small volume product that can be sold at a high price will be less dependent on process economics, though the profitability of the product will be adversely affected if process economics are unduly neglected.

It is also important to bear in mind that improvements to the productivity in the upstream process will not necessarily help the downstream process. The scale of the downstream process will not be likely to change, even though the cell culture vessel may be a fraction of the size previously planned - the amount of product being purified will not change. Indeed, it is possible that changes in the upstream process may be beneficial in terms of productivity, but if the impurity profile is also changed the downstream process will have to be adapted accordingly.

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