Affinity Chromatography: The Fine Print by Pete Gagnon, Validated Biosystems Affinity chr o m a t o g ra p h y is often held up effector functions. Antibodies are a good as the ideal in chr o m a t o g ra p h y. Exquisite example. The non antigen-binding parts of sp e c i f i c i t y . Unmatched simplicity. Load it, the molecule are richly endowed with recep- wash it, elute the purified product. Wh a t tors that interact with a variety of proteins, could be better? Affinity chr o m a t o g ra p h y ca r b o hy d r ates, and cells in the immune sys- could be better for one thing. There is a ver y tem. Conformational modification of these high price tag for both the specificity and the receptors can alter their functionality. All of si m p l i c i t y , and this isn’t referring to the dollar these factors — elevated tendency towar d cost for the media itself. Affinity chr o m a t o g - aggregation, proteolysis, and alteration of rap h y invol v es complications that have effector functions — can alter product effec- immensely important ramifications in purifi- tiv i t y , safety, and pharmacokinetics — and cation process development. The only thing this is before even taking into considerat i o n more costly than the complications them- that the elution conditions may have altered se l v es, is overlooking them. a protein’s primary therapeutic function. Pr oduct denatur at i o n . One of the most One of the ways to deal with product serious and persistent concerns about affinity al t e r ation is to develop milder elution condi- chr o m a t o g ra p h y is the potential for product tions. The first objection to this suggestion is de n a t u r ation. This is especially the case with usually that “If I use a less extreme pH, then strong affinity ligands that require harsh con- the elution will be less effective. ” In fact, elu- ditions for elution. The elution method of tion pH can often be raised without any loss choice is usually exposure to low pH, typical- of effectivi t y , and it’s certainly worth eval u a t - ly in the range of pH 2.5-3.0. Detailed studies ing a given application to find out what the of protein conformation under these condi- real limits are. If modifying pH alone doesn’t tions have documented permanent conforma- bear fruit, there are other opportunities. tional changes as a result of such exposure. Biological affinity interactions are mediated Hydrophobic residues normally protected in by complex combinations of hyd r o p h o b i c the interiors of the primary structural domains in t e r actions, charge interactions, hyd r o g e n become exposed on the surface, increasing bonding, and other mechanisms. Instead of the tendency for the protein to become using just low pH to twist a protein out of in vol v ed in nonspecific hydrophobic interac - shape so severely that it can’t remain bound tions. Elevated tendency toward aggregation to an affinity ligand, it is almost always possi- is one of the results, as is easily proven by ble to target one or more of the actual bind- examining size exclusion chr o m a t o g ra p h y ing mechanisms. This alone usually won ’ t be profiles following affinity purification. sufficient to cause elution, but it almost A usually less obvious but still common al w ays ameliorates the severity of the pH side effect of such changes is an elevat e d conditions required to elute the protein. For te n d e n c y toward proteolysis of the product. example, hydrophobic interactions can be This tends not to be noticed unless it’s weakened with up to 50% ethylene glycol. se v ere, but it’s common nevertheless. The Et h ylene glycol is actually stabilizing to most most insidious side effect — and the most proteins up to this level, but at the same time overlooked — is modification of secondary it is a very effective polarity reducer. It’s also 2 nonionic and won ’ t interfere with down - studied affinity interactions consistently stream charge-based purification methods. re v eal that when binding occurs, it is accom- Hydrogen bonds can be suspended by the panied by a phenomenon called induced fit. inclusion of 1.0M urea. At this concentrat i o n Induced fit refers to a situation where after it ’ s conformational effects on a protein are coming into contact with one another, either nil, and like ethylene glycol, it is nonionic. the affinity ligand, its receptor, or both, 0.5-1.0M NaCl will help to suspend any undergo conformational changes that lock charge interactions, without significant them into place. One of the best prac t i c a l enhancement of hydrophobic interac t i o n s . indicators that induced fit is occurring is The alternative use of a chaotropic salt like when you can bind a product to an affinity sodium perchl o r ate at the same concentrat i o n ligand under mild conditions, but very harsh will likewise suspend charge interactions and conditions are required to remove it. One of ma y further weaken the interaction through the best cha r acterized examples of this is the its chaotropic properties. binding of protein A to IgG, whi c h binds in Using combinations of these mecha n i s m s the hydrophobic cleft between the Cg2 and often makes it possible to raise elution pH by Cg3 domains. X-ray crystallographic data a full pH unit; sometimes substantially more. sh o ws that the Cg3 domain is unaffected by The question invariably arises: Don’t these the contact, but the adjacent Cg2 domain is complex formulations exert the same denatu- displaced longitudinally toward the protein A rat i ve effects as pH alone? They don’t. Thi n k and Cg3. Besides altering the local confor- of it like this. Let’s say that you have a partic- mation, this destabilizes the receptor-r i c h dis- ular oak board that you would like to remove tal third of the Cg2 domain, whi c h in turn from a wall so that you can use it for some- causes a partial rotation and destabilization thing else. It is screwed to the wall, nailed to of the carbohyd r ate domains between the the wall, and glued to the wall. If you just try Cg2 domains. The effect is apparently perma- to rip it off with a crow- b a r , your chances of nent. No matter how careful you are to devel - re c o vering it intact are grim. But if you op gentle elution conditions, comparison of re m o ve the screws and nails, then weaken the purified product against a non-affinity the glue by pouring hot water around the purified control virtually always confirms edges, you can remove it with much less me a s u r able changes in key behavi o r al fea- fo r ce, and your chances of recovering it tures of the product. This is not to say that intact are improved proportionately. This rai s - de v eloping mild elution conditions is not es the second objection to this approach: it wor t h w hile. Doing so can make an immense cancels out part of the simplicity of affinity difference. But it may not be able able to chr o m a t o g ra p h y. Library research will often avert denaturation problems entirely. lighten the load by revealing the dominant Le a ch i n g . The second major concern with me c hanisms of an affinity interaction, but affinity chr o m a t o g ra p h y is leaching of bioac- there will still need to be some experimenta- ti ve ligand and contaminants that may be tion to develop the most effective while least associated with it. This issue gets under- denaturing elution buffer. pl a yed but it is very serious. The inevitability In spite of taking great pains to develop a of leaching is the reason the FDA insists that nondenaturing elution buffer, you may still an y biological affinity ligand used in the find that your product has an elevated ten- manufacture of a biological product meet the de n c y to aggregate, or that it exhibits elevat - same application requirements as the end ed vulnerability to proteolysis, or that it product itself. This extends even to how the exhibits modified primary or secondary func- affinity ligand is purified. For example, the tions. The ugly truth is — to varying degrees protein A going into many chr o m a t o g ra p h y — that this is largely inevitable. The best products is purified by affinity chr o m a t o g r a- 3 ph y on immobilized human polyclonal IgG. se c o n d a r y , like complement fixation. Thi s The IgG column is potentially contaminated le a ves you with a high probability that with virus, whi c h can potentially leach into le a c hed affinity ligand will interfere with the purified protein A, and from there into either the direct function of your product, or your final product.