Page 13 - Subyek Encyclopedia - Encyclopedia of Separation Science

8 I / AFFINITY SEPARATION / Derivatization

sensitivity are maximized. In contrast, for preparative used to determine degradation of the target protein
and process applications, the objective is to maximize (for example deamidated and oxidized elements); to
purity, yield and economy. These techniques have identify previously unidentiRed components; to estab-
developed separately, simply because biological mac- lish the chromatographic identity between recom-
romolecules pose unique difRculties, making binant and natural materials; to develop orthogonal
them unsuitable for ‘standardized’ analysis. A major methods for the identiRcation of unresolved impu-
inSuence on this division has been that scale-up usu- rities; and for many other demanding analytical
ally occurs very much earlier in the development of approaches.
a process, causing biochemists to turn to the tradi-
tional low performance methods of ion exchange Af\nity versus Traditional Media
(IE), hydrophobic interaction (HI) and gel per-
meation chromatography (GPC). The highly efR- When projects are transferred from research to devel-
cient afRnity chromatography method was gener- opment two sets of chromatographic techniques are
ally ignored, primarily because of the difRculty of carried forward: analysis, usually based on RP-
having to develop a unique ligand for each separation HPLC; and larger scale serialized separation steps,
rather than having ‘off-the shelf’ column pack- often incorporating traditional methods of ion ex-
ings immediately available from external suppliers. change, hydrophobic interaction and gel permeation
For analytical purposes high performance afRnity chromatography. Major decisions have to be taken at
liquid chromatography (HPALC) is a rarity, a func- this juncture } to scale up the separation processes
tion of the limited availability of suitable matrices developed during the research phase or to investigate
(Table 2) and the afRnity process itself. alternatives. Regulatory demand and shortened pat-
Where quantiRable high speed chromatography is ent lifetimes compel managements to ‘fast track’ new
required, reversed-phase HPLC (RP-HPLC) has no products. Commercial pressure is at a maximum.
equal. Unfortunately there is no general purpose Being Rrst to market has the highest priority in terms
method for biomolecules to parallel the inherent of technical and commercial reward. Very little time
power of RP-HPLC. The success of RP-HPLC for is left to explore other separation strategies. It is
analysis can be judged from the large number of known that serial application of IE, HI and GPC
published applications developed for the ‘Rrst wave’ inevitably leads to very high manufacturing costs,
of protein pharmaceuticals manufactured by geneti- but which comes Rrst? Most often the decision is
cally engineered microorganisms. These extracellular taken to begin manufacture using unoptimized separ-
(relatively) low molecular weight proteins include ations as deRned in research reports. It is only in
human insulin, human growth hormone and the in- retrospect that very high production costs become
terferons. However, as molecular size and fragility apparent. By then it is too late } regulatory systems
increase, so difRculties in using HPLC increase, are Rrmly in place.
a primary reason why much analysis is still conducted There is an alternative. If researchers were more
on low performance systems. Low performance sys- aware of process economics and the consequences of
tems are easily scaled up; RP-HPLC is not. regulatory demand, selection of superior separation
Biological separation systems must be aseptically processes could then result. Although most resear-
clean throughout the process. The mixtures are inevi- chers are fully aware of the advantages of single-step
tably complex and usually contain many contamina- afRnity methods, paradoxically the high selectiv-
ting similarly structured species. Such species can ity advantage of afRnity chromatography is also a
adsorb very strongly onto the medium, demanding weakness. Suitable off-the-shelf afRnity adsor-
post-use washing with very powerful reagents to ster- bents are often unavailable, in which case an adsor-
ilize and simultaneously clean the column. Silica- bent has to be custom synthesized. Since the majority
based matrices cannot survive this type of treatment, of biochemists have no desire (or time) to undertake
hence scale-up of analytical procedures is generally elaborate chemical synthesis, antibody-based adsor-
precluded. The Rrst wave of commercial protein bents are commonly used. However, raising suitable
pharmaceuticals have generally proved to be relative- antibodies and purifying them before immobilization
ly stable under high stress conditions. On the other onto a preactivated support matrix is an extremely
hand intracellular proteins, often of high molecular laborious procedure. In addition, proteins are so of-
weight, are unstable. Analysis by high performance ten tightly bound to the antibody that subsequent
RP-HPLC methods then becomes problematic. De- elution involves some degree of denaturation and/or
mand for fast, high resolution, analytical methods loss of acitivity. Ideal media require the incorporation
will continue to increase for on-line monitoring and of elements of both nonselective and selective adsor-
process validation. Such techniques have already been bents to provide adsorbents with a general applicabil-