Affinity versus Traditional Media

When projects are transferred from research to development two sets of chromatographic techniques are carried forward: analysis, usually based on RP-HPLC; and larger scale serialized separation steps, often incorporating traditional methods of ion exchange, hydrophobic interaction and gel permeation chromatography. Major decisions have to be taken at this juncture - to scale up the separation processes developed during the research phase or to investigate alternatives. Regulatory demand and shortened patent lifetimes compel managements to 'fast track' new products. Commercial pressure is at a maximum. Being first to market has the highest priority in terms of technical and commercial reward. Very little time is left to explore other separation strategies. It is known that serial application of IE, HI and GPC inevitably leads to very high manufacturing costs, but which comes first? Most often the decision is taken to begin manufacture using unoptimized separations as defined in research reports. It is only in retrospect that very high production costs become apparent. By then it is too late - regulatory systems are firmly in place.

There is an alternative. If researchers were more aware of process economics and the consequences of regulatory demand, selection of superior separation processes could then result. Although most researchers are fully aware of the advantages of single-step affinity methods, paradoxically the high selectivity advantage of affinity chromatography is also a weakness. Suitable off-the-shelf affinity adsorbents are often unavailable, in which case an adsorbent has to be custom synthesized. Since the majority of biochemists have no desire (or time) to undertake elaborate chemical synthesis, antibody-based adsorbents are commonly used. However, raising suitable antibodies and purifying them before immobilization onto a preactivated support matrix is an extremely laborious procedure. In addition, proteins are so often tightly bound to the antibody that subsequent elution involves some degree of denaturation and/or loss of acitivity. Ideal media require the incorporation of elements of both nonselective and selective adsorbents to provide adsorbents with a general applicabil ity. If stable, highly selective and inexpensive affinity ligands were available, then opportunities would exist for researchers to develop efficient high yield separations even in the earliest phases of investigation. These systems could then be passed forward to production with the knowledge that optimally efficient separations are immediately achievable.

Production costs of any pure material reflect the absolute purity level required and the difficulty of achieving it. Therapeutic proteins have high purity requirements and the larger the administered dose, the purer it has to be. Since many protein pharmaceuticals will be used at high dose levels, purities need to exceed 99%, occasionally up to 99.999%. That these purities can be met by traditional methods is possible, but it is widely documented that the application of such methods massively increases production costs. Between 50 and 80% of total production costs of therapeutic proteins are incurred at the purification stage. The manufacturing cost of a product is directly related to its concentration in the mother liquor; the more dilute it is, the higher the cost of recovery. Since traditional purification processes on their own cannot selectively concentrate a target protein to the exclusion of all others, they have to be used in series. The number of stages required can vary between four and 15. Each step represents a yield loss, and incurs a processing cost. Yields of less than 20% are not uncommon. Figure 3 shows an enzyme purified in multiple stages and by a one-step affinity process.

It was these limitations that caused biochemists to examine highly selective ligands. Almost any compound can be used as an affinity ligand provided it can be chemically bonded onto a support matrix and, once immobilized, it retains its ability to interact with the protein to be purified. The ligand can be a simple synthesized entity or a high molecular weight protein. The affinity technique is theoretically of universal application and any protein can be separated whatever its structure and origin. As always, there are major limitations. The most effective affinity ligands are other proteins. Unfortunately such proteins are difficult to find, identify, isolate and purify. This results in high costs. An even greater deterrent is that most proteins are chemically, cata-lytically and enzymically unstable, a particularly unattractive feature if they are to be used for the manufacture of therapeutic substances; and regulatory authorities generally reject applications using pro-teinaceous ligands.

In anticipating that one day stable inexpensive affinity media would be in demand, a team led by C.R. Lowe began an investigation into which synthetic ligand structures offered the greatest possibility of developing inexpensive stable ligands. It was concluded that structures that could be manipulated into specific spatial geometries and to which intermolecular binding forces could easily be added offered the highest chance of success. Model compounds were already available; the textile dyes.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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