combinatorial chemistry

Current Pharmaceutical Biotechnology, 2011, 12, 000-000 1

1389-2010/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.

Capturing and Amplifying Impurities from Recombinant Therapeutic Proteins Via Combinatorial Peptide Libraries: a Proteomic Approach

Pier Giorgio Righetti*, Egisto Boschetti and Elisa Fasoli

Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Via Mancinelli

7, 20131 Milano, Italy

Abstract: The technique of combinatorial peptide ligand libraries (CPLL), for capturing and amplifying low-abundance

proteins in r-DNA products as well as in a number of other biological systems, is here analyzed in depth and reviewed.

This methodology is based on a creation of several millions of bio-specific ligands composed of hexapeptides produced in

a combinatorial way. When acting on an overloading and saturation principle, high-abundance species are captured in lim-

ited amounts, whereas low-abundance ones keep being concentrated on their bio-specific ligand till substantial harvesting

from solution (the capture process occurring in general from ca. 50% up to 90% efficiency). Examples are given on track-

ing host-cell impurities present in, e.g., recombinant albumin or monoclonal antibodies. Additionally, other examples of

detecting traces of additives and fining agents in such beverages as white and red wines are presented. The unique mecha-

nisms underlying the protein capture in the CPLL methodology, as opposed to capture by homogeneous beads, as repre-

sented by ion-exchangers and by hydrophobic resins, are discussed in depth.

Keywords: Combinatorial peptide ligand libraries, host protein contaminants, low-abundance proteome, trace impurities, rDNA proteins.

INTRODUCTION

A number of biopharmaceuticals today in use are prod-ucts from recombinant DNA technology or derived from human plasma. Recombinant proteins are expressed in se-lected host cells under controlled conditions while human plasma derived products are extracted from pooled human plasma. Both are complex starting materials comprising thousands of proteins which are potential impurities for the final product that may, in some cases, cause adverse events in the patient ranging from fever to long-term immunogenic-ity to toxic and, in rarer cases, fatal events. Host cells used for the biosynthesis of recombinant proteins are relatively complex systems extending from bacteria (e.g. Escherichia coli) [1-5], to yeasts (e.g. Pichia pastoris) [6-10] and to mammalian cells such as Chinese Hamster Ovarian cells (CHO) [11]. During culture, these cells secrete a very large number of their own proteins that can easily contaminate the recombinant DNA product [12-16]. Even after sophisticated purification steps, significant levels of host cell proteins (HCP) may still remain present in the final purified biophar-maceutical [17-23]. Although host cell impurities could be innocuous to the receiving patient, regulatory agencies re-quire demonstrations that HCPs are not only minimized but also analyzed with the most sensitive available methods. Current analytical methods are limited in number and also not sufficiently sensitive for the detection of trace levels of HCPs. Current HPLC techniques have good resolution; how-ever, they suffer from their low sensitivity, the possibility of non-specific binding and are of subjective interpretation.

*Address correspondence to this author at the Department of Chemistry,

Materials and Chemical Engineering “Giulio Natta”, Via Mancinelli 7, Politecnico di Milano, 20131 Milano, Italy; Tel: +39-02-23993045; Fax:

+39-02-23993080; E-mail: [email protected]

Electrophoretic analytical methods (e.g. SDS-PAGE with silver staining) also offer good resolution, but sensitivity is low and the interpretation remains very subjective as well. Immunological determinations are more specific than HPLC and electrophoresis; however, they suffer from varied affini-ties of the selected antibodies. Moreover they may not be reproducible from lot to lot, especially when very low con-centration species must be detected. In addition detection of HCPs is only possible if the whole antigen repertoire is cov-ered by antibodies. If antibodies to a specific antigen are missing the impurity simply escapes detection. Immunode-tection can be separated into two groups: Western blots and Immunoassays. The former has a sensitivity of about 10 ppm and the latter a sensitivity lower than 1 ppm. In conclusion, all detection methods for HCPs face a challenging problem: how to deal with very low concentrations of contaminant proteins present in “pure” biopharmaceuticals after separa-tion / purification with current processing techniques.

A few years ago, we reported a novel approach for cap-turing the "hidden proteome", i.e. those rare and very rare proteins that constitute the vast majority in any cell or tissue lysate and in biological fluids [24-30]. It is based on a com-binatorial library of hexameric peptide ligands (CPLL) bound to porous beads. Each bead contains a single hexapep-tide sequence distributed throughout its porous structure reaching concentrations of about 50 pmoles per bead. This amounts to a ligand density of ca. 40-60 moles per mL of bead volume (average bead diameter of about 70 m). Thus each single bead has millions of copies of a single, unique ligand and each bead potentially has a different ligand from every other bead. If for the synthesis the 20 natural amino acids were used the library should contain a population of linear hexapeptides amounting to 20

6, i.e. 64 million differ-

wasim

Final

2 Current Pharmaceutical Biotechnology, 2011, Vol. 12, No. 12 Righetti et al.

ent ligands. With such vast populations of heterogeneous ligands, in principle, an appropriate volume of beads could contain a partner able to interact with just about any protein present in a complex proteome (be it a biological fluid or a tissue or cell lysate of any origin). This ligand library has been efficiently used for capturing and revealing a very large population of previously undetected proteins in urine [27], serum [31], red blood cells [32], cerebrospinal fluid [33], egg white [34] and yolk [35] as well as cow’s whey [36], venoms [37, 38] and minor latex protein allergens [39].

Although the examples reported above regard classical proteomic approaches, purification of proteins and identifica-tion of new compounds, another important application is that of capturing and "amplifying" trace amounts of impurities present in r-DNA proteins available on the market and meant for human consumption. Several recombinant products for human therapeutic use have been already approved by FDA and are currently in the market, such as human insulin, choriogonadotropin, follicle-stimulating and luteinizing hormones, somatotropin, erythropoietin, human blood co-agulation factors (factors VII, VIII, and IX), interferons (a, b, and g) and interleukins, to name just a few [40]. Characteri-zation of a biotechnological or biological product (which includes the determination of physicochemical properties, biological activity, immunochemical properties, purity, and impurities) by appropriate techniques is important to allow relevant specifications to be enacted. Acceptance criteria should be established and justified based on data obtained from lots used in preclinical and/or clinical studies, data from lots used for the demonstration of manufacturing con-sistency, data from stability studies, and relevant develop-ment data. An inherent degree of structural heterogeneity occurs in r-DNA proteins due to the biosynthetic processes used by living organisms to produce them; therefore, the desired product can be a mixture of anticipated post-translationally modified forms (e.g. glycoforms). These forms may be active and their presence may have no delete-rious effects on the safety and efficacy of the product. The manufacturer should define the pattern of heterogeneity of the desired product and demonstrate consistency with that of the lots used in preclinical and clinical studies. Heterogeneity in r-DNA proteins can also be produced during their manu-facture and/or storage. Since the heterogeneity of these prod-ucts defines their quality, the degree and profile of this het-erogeneity should be characterized to assure lot-to-lot consis-tency.

Given all the complex problems outlined above, the ap-proach of exploiting the CPLL beads for r-DNA product analysis would have a double advantage: on the one side it would enable obtaining sufficient amounts of any potential impurities to allow for their chemical characterization; on the other side, it would permit purifying r-DNA products to an extreme degree, not achievable with present-day technolo-gies. We have indeed reported such analyses in various in-stances [41-43] and we here review some of the data ob-tained. Moreover, we will offer additional examples of track-ing trace additives in food stuff, permitting the observation of such minute amounts as to go undetected by any of the presently-available techniques. The results we will discuss suggest that regulatory agencies should add the CPLL meth-odology to the arsenal of screening/analytical methods today

extensively described and applied to a number of biological samples.

TRACKING HOST CELL PROTEINS IN r-DNA

PRODUCTS

Combinatorial peptide ligand libraries have initially been described and used as a means to concomitantly reduce the concentration of high abundance proteins in a sample while increasing the concentration of rare species in view of detect-ing and identifying very dilute gene products. Considering that in recombinant-DNA biologicals the target proteins are by definition of high-abundance and impurities of low-abundance (at least at the semifinal purified step), the com-parison with the primary application of CPLLs became quite obvious. Combinatorial peptide ligand beads were thus con-sidered as a new tool for either enhancing the level of impu-rity concentration to better detect them or eliminate protein impurities in view of obtaining a further purified material. For the first objective the CPLLs would have to be used un-der large overloading while for the second application a loading below the saturation of the packed bed by protein impurities would have to be mandatory. Both approaches have been experienced with interesting results [41-43].

In a first instance [42], recombinant human albumin, ex-

pressed in Pichia pastoris was investigated. This product

was commercially supplied as a 95% pure product after clas-sical purification processing. As shown in Fig. (1), when

analyzed by two-dimensional (2D) mapping, the purity of the

commercial product seemed relatively modest (panel A) since the expected presence of a major spot (see arrow),

clearly representing albumin, it is accompanied by a set of

five minor bands with identical pIs but progressively higher Mr values, indicating an oligomeric series (dimers, trimers

etc.). Moreover a number of other detectable impurities were

present as a series of minor spots of identical Mr and slightly higher pI (just to the right of the main albumin band) and a

more acidic spot of lower Mr value. The enhancement of

impurity traces by the treatment of the initial albumin sample by CPLLs evidenced a quite different picture. As shown in

panel B, which represents the eluate from the ligand library,

many more protein spots are visible, scattered in the 2D plane. A total count after background cleaning and streak

subtraction, performed with the PDQuest software, has re-

vealed more than 50 polypeptide chains, with the intact al-bumin (spot No. 1) now representing less than 50% of the

desired, pharmaceutical product. In order to ascertain the

identity of this large number of polypeptide chains, 26 of the visible spots in the 2D map of panel B were excised, di-

gested and analyzed by mass spectrometry. It would appear

that, indeed, most of these spots represent albumin frag-ments, ranging in mass from the native (Mr 71317) species

to as low as ca. 8000 Da, as assessed from the SDS dimen-

sion of the 2D gel. The large number of fragments observed may be a result of cleavages by proteases probably massively

present in the culture media produced by the yeast itself. A

number of Pichia pastoris proteins have also been identified among the spots analyzed. Here too it is of interest to note

that, in the control, essentially none of these spots could be

detected, except for the large albumin fragment with a mass of ca. 50 kDa (spot No. 2 in panel B).

Proteomics of rDNA Proteins Current Pharmaceutical Biotechnology, 2011, Vol. 12, No. 12 3

To be considered as a pure product this recombinant hu-man albumin should at least be free of all host proteins and possibly depolluted of albumin fragments and of oligomers, a task that appears to be reachable with the use of CPLLs. Actually the same recombinant albumin was treated with a CPLL-filled column. Fractions from the column were col-lected while progressively loading to ensure that the binding capacity level for impurities was not reached. Since the li-brary was able to capture also albumin, a little amount of it, estimated at about 1%, was lost, but impurities were re-moved quite efficiently as reported [41].

Similarly, a second protein, injectable human serum al-bumin, supplied as 96-98% pure solution, has been analyzed before and after impurity “amplification” with the hexapep-tide combinatorial library beads. SDS-PAGE analysis dem-onstrated the apparent purity of the product Fig. (2, track c), in which the only other “impurity” present was haptoglobin. However, following the CPLL treatment, many additional protein traces were observed, representing a number of major to medium-abundance serum species (track d). It was hy-pothesized that, if one could have treated a much higher sample amount, he might have been able to detect an impres-sive number of additional serum proteins that are unavoid-ably present at undetectable level.

A more challenging situation was the detection of impu-rity traces from recombinant very pure Protein A expressed in E. coli (purity estimated of 99.9%) [42]. With a relatively massive amount of this protein (about 4 grams of highly pu-rified material) it was possible to identify non only a number of Protein A fragments and aggregates, but more importantly many E. coli proteins that were not supposed to be present at all.

In another instance, our group analyzed by the peptide library technology monoclonal antibodies produced by r-DNA techniques [43]. Throughout the progression of mono-clonal drug development there have been four major anti-

body types developed: murine, chimeric, humanised and human. Chimeric and humanized antibodies have generally replaced murine antibodies in modern therapeutic antibody applications. Monoclonal antibody therapy is the use of monoclonal antibodies (mAb) to specifically bind to target cells. This may then stimulate the patient's immune system to attack those cells. It is possible to create a mAb specific to almost any extracellular/ cell surface target, and thus there is a large amount of research and development currently being undergone to create monoclonals for numerous serious dis-eases (such as rheumatoid arthritis, multiple sclerosis and different types of cancers). There are a number of ways that mAbs can be used for therapy. For example: mAb therapy can be used to destroy malignant tumour cells and prevent tumour growth by blocking specific cell receptors [44]. Variations also exist within this treatment, e.g. radio-immunotherapy, where a radioactive dose localizes on target cell line, delivering lethal chemical doses to the target. Re-cently the CPLL technique was applied to the analysis of residual impurities in purified monoclonal antibodies from hybridoma cell cultures grown in a basal medium supple-mented with bovine albumin and insulin, and human trans-ferring [43]. As shown in Fig. (3A), this preparation seems to be highly purified, since, in addition to the 25 and 50 kDa polypeptides, representing IgG subunits, and some oli-gomeric species for the heavy chains (marked by arrows); only a few extra spots can be appreciated, especially in the 10 to 20 kDa region. However, after peptide library treat-ment Fig. (3B) many new spots can be visualized, in the same 10-20 kDa space as well as in between the light and heavy IgG chains. This can be further appreciated in Fig. (3C), where the crosses mark the >70 spots excised from the gel and subjected to MS analysis [43]. These were found to be composed mainly of three classes of macromolecules: those derived from proteins present in the culture broth (no-tably albumin and transferrin), fragments of the desired final product, covering Mr ranges from as low as 5 up to 45 kDa, and some aggregates of light and heavy chains of IgGs

Fig. (1). Two dimensional electrophoretic maps of native purified recombinant human albumin (95% purity) (A) and host cell protein impu-

rities extracted using the combinatorial peptide ligand library (B). The pH gradient was from 3.0 to 10.0. Impurities are extended over a large

zone of isoelectric points and molecular masses. The arrow in panel A indicates the albumin zone. The numbers in panel B indicate the spots

eluted and subjected to MS analysis (from Fortis et al. see ref. 41, by permission).


(mostly dimers and trimers). From the data so far discussed, it would appear that most of the r-DNA compounds available on the market for human therapy are plagued by two main classes of impurities: (a) fragments of the desired therapeutic compounds, likely produced during storage by residual con-taminant proteases and (b) traces of several host proteins and proteins added to culture broths. Such levels of impurities are in general below current detection limits but are so highly “amplified” by the CPLL treatment that the Biotech Industry and Regulatory Agencies will have to take that into account.

TRACES OF PROTEINACEOUS ADDITIVES IN

FOOD STUFF

Although such a topic might appear not to be related to a journal of pharmaceutical biotechnology, in reality modern science believes that food is strictly related to pharmacology, as most of what we eat can affect, positively or negatively, our health status. Plenty of food stuff today is manipulated and supplemented with additives that could have a direct impact on our health. A case in point is represented by wines. These beverages are coming under close scrutiny from the European Community (EC), which is issuing rules in regard to their proteinaceous content. The reason is that, since several years, wine producers have been adopting fin-ing agents, aimed at reducing or totally eliminating potential sediments forming upon long-term storage in marketing out-lets. Although such sediments are not noxious at all, since they are composed mainly of residual grape proteins that flocculate upon standing, they are regarded with suspect by customers, who believe that the content of such wine bottles has spoiled. In order to eliminate these issues, wine makers

have introduced different types of fining agents that seem to be adopted the world over. In the case of white wines, the classical fining process involves addition of bovine casein, which forms complexes with residual grape proteins and induces massive flocculation. A second type of treatment, involving mostly red wines, is addition of egg albumin, which serves the same purposes. Although in principle there would be nothing wrong with these procedures, in practice there are issues of human health, since both these additives are well-known allergens, which could induce adverse reac-tions on sensitive people. Aware of that, the European Community has issued Directive 2007/68/EC, which states: “any substance used in production of a foodstuff and still present in the finished product” must be declared in the label, especially if it originates from allergenic material. Such rule should be implemented by this summer. Apparently, no wine producer has taken any action concerning this regulation, since, in most cases, after addition of casein or egg albumin, a second treatment with bentonite is carried out, supposedly removing any residual initial fining agent. Thus wine pro-ducers do not declare anything at all to the EC authorities simply because they believe (or have no way to detect) that no residual, potentially harmful proteins are still present in their products. But is this really the case? Apparently not. In a recent investigation, Paschke’s group [45] has indeed proved that - and -caseins remain in some white wines and are detectable, although their concentration has been esti-mated to be of the order of 0.1 to 0.2 mg/L. In another, re-cent report, Wigand et al. [46] have performed a similar kind of study on red wines, in comparison with rosé and white wines, just to detect any residual grape protein remaining in such products. They found that, indeed, in most of these

Fig. (2). Sodium dodecyl sulphate electrophoresis of injectable pure human albumin from serum (lane c) and captured proteins by the ligand

library (lane d). The table indicates the list of impurities found respectively in the initial sample and in the sample where impurity traces have

been amplified. Lane a: ladder of standard protein masses. Lane b: void. The arrow indicates the positioning of albumin (from Fortis et al.

see ref. 41, by permission)


Fig. (3). 2-D patterns of mAbs purified with a MEP-HyperCel resin. (A) Control (untreated) antibodies; (B) eluate from the CPLL beads; (C)

crosses marking the new spots detected (>70) after CPLL processing and subjected to MS identification (modified from Antonioli et al., see

ref. 43).


wines, some twelve residual grape proteins and six derived

from yeast could still be detected. Among them, the ubiqui-

tous allergen lipid transfer protein (LTP) as well as thau-matin-like proteins (TLP). Although the main aim of their

research was not the finding of fining agents, nevertheless

they also tested such wines for the presence of casein or egg albumin by ELISA tests. Their conclusions: “In both cases

the results were below the limit of quantification of the test

systems; no allergenic proteins have been found. Using mass spectrometry, we were not able to detect the presence of fin-

ing agent proteins in all the bands analyzed” [46]. Here

again, the use of the CPLL methodology has deeply ex-panded the horizon. In a report just issued, we have applied

CPLLs for detecting traces of casein in white wines, with

remarkable results: not only we could track down much lower amounts of casein than in [45], down to as little as 1

g/L (thus with an increment of sensitivity by at least two

orders of magnitude, see Fig. (4), but we could also prove, for the first time, that the signal amplification factor of

CPLLs could easily reach four orders of magnitude [47]. But

there is even more to be stated in this regard. Just as our pa-per [47] appeared at the web site of J. Proteomics, another

one was posted at the web site of J. Chromatography A [48]:

in this last paper, the authors state “when fined wine samples were considered, the lowest added concentration for which

the peptide marker could be detected was 50 μg/mL” (the

peptide marker referring to casein digests, as identified by mass spectrometry) [48]. Now, if we are not mistaken, this

means that our CPLL treatment for harvesting and detecting

minute traces of caseins in white wines has a sensitivity 50000 times better than the MS method of Monaci et al. [48]

(it goes without saying that we too identify the captured ca-

seins via MS). Curiously, as we applied CPLLs also to the analysis of Italian red wines, thinking that, in case of fining,

we would have found traces of egg albumin, much to our

surprise we found that such wines were exclusively treated with casein as well [49]. Here too, the lower sensitivity limit

of 1 g/L of added casein was easily reached. In addition,

and unexpectedly since nobody reported these findings so far, we also found traces of proteins from Saccharomyces

cerevisiae and a few proteins from plant pathogens and fungi

(e.g., Botryotinia fuckeliana, Sclerotinia sclerotiorum, As-pergillus aculeatus). In an additional project, we also ana-

lyzed the beer proteome via CPLL capture. Via mass spec-

trometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could

categorize the residual proteins present into 20 different

barley protein families and 2 maize proteins, the only ones that had survived the brewing process (the most abundant

ones being Z-serpins and lipid transfer proteins). In addition

to those, no less than 40 unique gene products were identified from Saccharomyces cerevisiae, as routinely used

in the malting process [50]. These latter species must

represent trace components, as in previous proteome investigations barely two such yeast proteins could be

detected. From all those examples, we can safely conclude

that the CPLL technology has much to offer in trace protein detection by a global proteome analysis and perhaps

represents the only tool that can dig to such a depth for

revealing minimal traces of species largely undetectable with present-day methodologies.

Fig. (4). SDS-PAGE of casein standards and a casein eluate from CPLL beads in a wine-like mixture consisting of 12.5% ethanol in water at pH 3.3 (with acetic acid) added with 1

g/L of casein. Mr: molecular mass standards; tracks 1, 2: 1 and 2 g casein standards treated in conventional Laemmli buffer; track 3: casein recovered from the wine-like mixture. Detection by silver staining (modified from ref. 47).

CPLLs IN THEIR INTIMATE INTERACTION

MECHANISM

Although CPLLs are now increasingly used in several laboratories in proteomic studies (as the beads have now been commercialized under the trade name of ProteoMiner from Bio-Rad Laboratories, Hercules, CA, USA) with excel-lent results (as amply reviewed by Righetti et al.) [51], the quite complex mechanism of action is sometimes subject to approximate understanding and also to misinterpretations; therefore it requires here some explanations. A recent review assembled most of what is known and collected from ex-perimental data [52]. To keep the explanation simple, CPLLs is a collection of mixed-mode sorbents used all together as a mixed bed. The mixed-mode properties of the peptide ligands are an extremely important feature that is at the basis of the interaction. Amino acids composing the peptide ligand can carry different pendant groups: some are mildly hydro-phobic (e.g. Ile, Leu, Val, Phe) others are ionic (e.g. Asp, Glu, Arg, Lys, His) and others are heterocyclic with charge transfer properties. In addition, these peptides display an intensive property to induce hydrogen bonding interactions. Most generally, when adding together hydrogen bonding, ion exchange effect and dipole interactions, ionic associations are the dominant interactions between a given protein and its partner hexapeptide, followed by hydrophobic associations. Interestingly, all of them might be present within the same


peptide ligand depending on the composition. Protein-protein (or protein-peptide) interactions are driven by the sum of a number of linkages, notably ion-ion, hydrophobic, hydrogen bonding and Van der Waals interactions, but only ion-to-ion are known as long-range interactions, in that they can occur at much longer distances in solution as compared to all other ones [67]. Given the nature of the library of peptide ligands, where ionic and hydrophilic residues are more abundant than hydrophobic ones, it is reasonable to expect that, if anything, ion-to-ion will be the first interactions to occur in the binding event among the hexapeptide baits and the native protein in a physiological solution. As a proof of that, it has been re-cently reported that the CPLL capture is strongly pH-dependent [53], to the point that it is advised to perform pro-tein capture at 3 different pH values, pH 4.0, 7.2 and 9.3 when attempting to get the largest population of low abun-dance species, since, outside large overlapping, different protein populations are harvested at these pH values. In addi-tion more than half of captured proteins are desorbed by in-creasing the ionic strength which is on the contrary known for its capability to enhance hydrophobic associations.

When considering that very many peptides are present in CPLLs under a mixed bed configuration, the situation be-come quite complex with a large number of displacement effects. This physicochemical reality contrasts fundamentally with the recently interpretation made by Keidel et al. [54]. It has in fact been indicated that the interaction of CPLLs with proteins is fundamentally hydrophobic with other very minor interactions to the point that ProteoMiner beads were com-pared with chromatographic beads exhibiting single-mode hydrophobic surface chemistries, such as Sepabeads FP-OD400 octadecyl, FP-DA400 diethylamine, FP-BU400 bu-tyl. Hexapeptide libraries would not induce large selectivity and the protein capture could “be explained by a general hydrophobic interaction mechanism, where diversity in sur-face ligands plays only a negligible role”. If this interpreta-tion were correct the use of peptide ligand library would not be adoptable for the enhancement and the separation of im-purities from biologicals as described in the previous section. If the interaction were hydrophobic the adsorption properties would not be applicable for generalized polishing application for three reasons: first they are not mixed-mode ligands, sec-ondly they are not used in mixed-bed configurations and thirdly they have never been used in overloading situations, three main conditions that need to coexist to catch and/or enhance the residual impurities from pre-purified biologicals. As a whole it is to be noticed that CPLL is a global mixed-bed approach to generalize the largest capture of very many distinct proteins than any other chromatographic approach at a point that from this same library it has been possible to sort out a number of specific peptide ligands for their highly se-lective extraction of given proteins from a variety of biologi-cal extracts (see for example refs. 55 and 56).

A library is a collection of beads each of them carrying a different anchoring chemical structure combinatorial or not. In the present case the ligands are peptides and each bead carries a different peptide under a quite high grafted density. Thus each bead is entitled to adsorb a different analyte; how-ever, due to some peptide affinity similarities for a given protein analyte, it may not be "yes-no" but modulated by (i) the respective concentrations of analytes, the conditions of

interactions and the similarity of protein epitopes. This pecu-liar situation calls for extensive protein displacement effects. The latter is never the case when dealing with homogeneous sorbents such as ion-exchange media or hydrophobic sorb-ents where all the beads are absolutely identical. The result-ing saturation effects of these two very different sorbents is also much differentiated. In library sorbents the saturation is played at each bead level for each individual protein com-posing the mixture. As explained in a recent review [52], the larger the sample loaded for a given volume of beads the larger the number of low-abundance proteins detected. His-torically the ligand library beads were designed for the iden-tification of hexapeptide structures that were thus used for the purification of one protein of the mixture. In other words, once the peptide ligand was identified, it was used as an af-finity column where only (or mostly) the target protein was captured while all other proteins were eliminated in the flowthrough. A large number of papers have been published on this technology since the beginning of the 90's and few of them referenced here [57-62]. From a homogeneous sorbent it is clearly impossible to make this selection since all the beads are identical and by definition they work exactly simi-larly. Already in the year 2000 Kay et al. [63] postulated that short peptides could be used for protein-protein interaction studies when they demonstrated that a number of peptides could interact specifically with some proteins. It was envi-sioned that to map interactions peptide ligands would be isolated from libraries and that interactions would accelerate functional analysis of proteomes and drug discovery. At the light of all these argumentations, it would appear that the retention mechanism proposed by Keidel et al. [54] is not tenable.

TRADITIONAL IMPURITY REMOVAL BY SOLID

PHASE EXTRACTION

Solid-phase extraction (SPE) and polishing, a final stage of eliminating impurities that are still present even after or-thogonal multistep procedures, have always been considered key steps in the preparation of very pure biologicals. Consid-ering the low amount of impurities, but under a possible very large diversity of proteins, several approaches have been suggested, but so far none of them are neither ideal nor of general application. Solid-phase extraction (SPE) is a separation process by which compounds, that are dissolved or suspended in a liquid mixture, are separated from other compounds in the mixture according to their physical and chemical properties. Analytical laboratories use SPE to con-centrate and purify samples for analysis. SPE can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue [64]. SPE uses the affinity of solutes dissolved or sus-pended in a liquid (mobile phase) for a solid through which the sample is passed (stationary phase) to separate a mixture into desired and undesired components. The result is that either the desired analytes of interest or undesired impurities in the sample are retained on the stationary phase. The por-tion that passes through the stationary phase is collected or discarded, depending on whether it contains the desired ana-lytes or undesired impurities. If the portion retained on the stationary phase includes the desired analytes, they can then be removed from the stationary phase for collection in an


additional step, in which the stationary phase is rinsed with an appropriate eluent. This technique can be very effective, even when the solutes are present at extremely dilute concen-trations (e.g. ppb). Materials extracted in this way can be used for subsequent chromatographic separation, spectro-scopic examination, or biological assessment. The apparatus consists of a simple tube, which may be 2-4 mm I.D. and 2-4 cm long and is, usually, made from stainless steel or a suita-bly inert polymer. The extraction tube is usually packed with an appropriate bonded phase. The most effective for extract-ing dispersive materials (e.g. hydrocarbons, chlorinated hy-drocarbons, pesticides, etc.) from water being long chain hydrocarbon bonded phases (e.g., a C18 phase). This system can extract many liters of sample if necessary and all the solutes accumulate at the front of the packing. The solutes can then be displaced from the adsorbent by 0.5 to 1 mL of methanol or acetonitrile (usually by reversed-flow tech-niques to minimize extract dilution). The result would indi-cate a concentration factor of 2000-4000. Providing the right adsorbent is chosen, the extraction process can be very effi-cient, e.g. 99%. The choice of adsorbent can be quite critical.

Outside CPLLs used as polishing tool other procedures based on solid-phase adsorption were described, but still they are much less global than CPLL. They use the regular prop-erties of chromatography sorbents such as hydrophobic ad-sorbents, ion exchangers, gel filtration media and various capturing ligands as phenyl boronates. In the domain of pol-ishing, the approach that gives some interesting results is based on strong anion exchange chromatography [65]. Here the pH of separation is adjusted in such a way so that the main protein has the same global charge of the solid phase and is consequently not adsorbed while some or most impu-rities have a charge complementary to the solid phase media and are therefore eliminated. In spite of interesting results, this process suffers from the fact that impurities that have the same or similar isoelectric point of the main protein are not eliminated.

Reversed-phase chromatography is definitely the strong-est case of any hydrophobic interaction [66]. It consists of a bonded phase comprising a long hydrocarbon chain (C18) at high ligand density. This C18 resin does not bind well pro-teins because of the presence of residual ionic charges on the surface of the sorbent unless using “ion-paring” agents. In this case the resin adsorbs quite tenaciously to the protein surface, but does not make any distinctions between the tar-get protein and impurities. This is simply because, contrary to what happens with CPLLs, the phenomenon of individual beads selectivity associated with the oversaturation is absent. Hydrophobic interaction chromatography (HIC) [67] is milder compared to RPC and does not normally comprise residual ionic charges. The density of the hydrophobic ligand on the bead surfaces is considerably less; additionally, such ligands are in general less hydrophobic and are thus made with shorter hydrocarbon chains, such as butyl to octyl. Un-der these circumstances protein can be captured when mixed with large amounts of anions and cations that follow the well-known Hofmeister (lyotropic) series. HIC has been adapted for polishing processes [68] even at large scale. Physicochemical conditions have been devised to have the major protein in the flowthrough while impurities would be randomly captured by the beads. Also in this case the impu-

rity removal is relative to hydrophobic polypeptides and not to all protein impurities. As described for anion exchange chromatography, physicochemical conditions have to be optimized case by case to have the major protein in the flowthrough, which means that all protein impurities similar to the target proteins in terms of hydrophobic degree are still present. Gel filtration, capable to discriminate proteins by their molecular mass, has also been described as a mean of removal of last impurities in a final polishing step. For in-stance this procedure has been reported for the polishing of a recombinant kinesin [69]; the final gel filtration process was able to increase the purity of the final protein from 96 to 99%. Nevertheless gel filtration is ineffective to remove im-purities that have a mass similar to the target protein.

Grafted aminophenylboronate ligand on chromatographic beads was also described as a solid support for polishing purposes [70]. However, this sorbent addressed different impurities to remove, such as lipopolysaccharides and DNA traces, both also frequently present as last impurities to be eliminated. All the above fundamental differences between single-mode adsorbents used in homogeneous mode and mixed-mode sorbents used in mixed-bed columns requires some more detailed explanations (see Table 1). Libraries of ligands based on their diversity of recognition addressing most if not all protein impurities at a time are the sole possi-bility of polishing and identification by a global proteomic approach. Given the above conditions and limits, it would appear that our CPLL methodology might be a particular case of SPE: although some similarities are there, indeed CPLLs represent a novel technology that has little to share with SPE. To start with, SPE per se does not contain any novelty in respect to the array of techniques available in Separation Science, as nicely summarized in the classical book by Giddings [71]. Basically, solid phase extraction car-tridges and disks are available with a variety of stationary phases, each of which can separate analytes according to different chemical properties. Most stationary phases are based on silica that has been bonded to a specific functional group. Some of these functional groups include hydrocarbon chains of variable length (for reversed-phase SPE), quater-nary ammonium or amino groups (for anion exchange) and sulphonic acid or carboxyl groups (for cation exchange). Thus, SPE just exploits well-ingrained chromatographic principles that have been operative in Separation Science since decades. As such, SPE acts on general adsorption prin-ciples, not targeted to specific biological activities, but rather to general properties of molecules, such as their hydropho-bicity, surface charge, etc. Moreover, SPE is largely adopted for adsorption of small organic molecules and analytes. Con-versely, the CPLL methodology is based on a biological rec-ognition principle, i.e. specific binding of a hexapeptide (the bait grafted onto the CPLL beads) to a complementary amino acid sequence onto he surface of a native macromolecule. Thus, the CPLL method resembles more affinity chromatog-raphy [72] or even antigen antibody recognition principles. We do share, however, with SPE, the unique amplification factor of the harvested analytes: in the case of SPE it is stated to be at least 3 orders of magnitude; in CPLLs, it seems that we can reach quite well four orders of magnitude. The CPLL capture principle will be described in more detail below.


Table 1. Properties and Capabilities of Various Methods to Remove Protein Impurity Traces

Method Impurity Addressed Initial Conditions LIGAND TYPE Bed Type

CPLL All All Mixed-mode Mixed bed

RPC Highly hydrophobic Solvent mixture Single-mixed mode Homogeneous bed

HIC Hydrophobic Lyotropic salts Single mode Homogeneous bed

IEX Cationic or anionic pH & ionic strength Single mode Homogeneous bed

GF Low masses All No ligand Homogeneous bed

Boronate DNA & LPS All Mixed mode Homogeneous bed

SPE Various Various Mixed mode Homogeneous bed

The limitations of the reported technology are currently well known. The first is a thermodynamic limitation: the capture of individual impurities would be reduced to zero when the affinity constant of the analyte for the beads is lower than the concentration of the analyte itself. The second is that, in spite of the extreme large spectra of ligands, some proteins are still looking for their own ligand partner. Never-theless that situation has been impressively improved with the use of carboxylated libraries, on one side [51] or the use of the current peptide library at different pH values so as to modulate the affinity constants for more adapted interactions [53], on the other side. These approaches are evidently appli-cable to the detection of recombinant protein impurities. The use of ligand libraries allows enhancing quite significantly the detectability of very-low abundance species; however, it does not influence the analytical determinations that are cur-rently used. At most they are to be adapted case by case. For instance immunoassays, involving the proper recognition of enhanced proteins, may require to neutralize the library elu-ate or to remove agents preventing the antigen-antibody re-action to occur.

CONCLUSION & PROSPECTS

For the first time with solid-phase combinatorial ligand library the question of resolving the universal polishing steps has been approached. The technology virtually applies to any possible purified protein from any possible initial crude ex-tract. Given the large diversity of mixed-mode ligands within a mixed-bed confined volume the technology can easily be applicable to solutions of different ionic strength and pHs without preliminary adjustments with significant advantages of direct streamlining with the preceding purification step. The use at large overloading conditions is here easy to apply because the quite large availability of samples with conse-quent large enhancement of low-abundance species. This point is of utmost importance when dealing with additives not only in food and beverages as underlined in the text, but also when dealing with vaccines made for instance on eggs. The production of human vaccines as for instance the influ-ence vaccine and measles-mumps-rubella vaccine is obtained with viruses that are propagated in embryonated eggs. Once the production process is completed egg proteins are re-moved. Unfortunately the final vaccinal preparation contains residual egg proteins, among which ovalbumin is probably

the most representative due to its highest abundance in egg white. In a number of cases allergic reactions to patients against egg proteins have been recorded to the point that recommendations are made not to eat eggs for several days after vaccination. This represents a high-risk concern for individuals including asthmatic patients [73]. Ovalbumin, whose trace content is variable from a producer to another, seems responsible for adverse effects [74]. However, other proteins could presumably be present with larger immuno-genic reactivity. Therefore checking for the presence of for-eign protein traces with highly sensitive approaches appears as being a major point.

An emerging field of concern in agrifood is the develop-ment of genetically-modified organisms (GMO) especially vegetables intended for human food. A way to check the action of the introduced new gene is to check its protein ex-pression. Beyond the information allowing the detection of GMOs, it allows identifying the protein expressed and its biological activity with its possible effect. CPLLs here also would contribute to elucidate the question as a result of their ability to enhance low-abundance species of any origin and finally to identify them.

Naturally the described technology is still at its initial stage and probably requires some more refinement and un-derstanding. For instance it is not fully clear if isoforms or post translational modifications can be highlighted. Based on the peptide-protein recognition mechanism, PTM such as glycosylation or phosphorylation should not interfere with the recognition mechanism. Nevertheless these modifications may change locally the property of protein epitope with some influence on the dissociation constant. These points need to be investigated further.

ACKNOWLEDGEMENTS

PGR gratefully acknowledges support by Fondazione Cariplo (Milano) and by PRIN-2008 (Rome).

ABBREVIATIONS

HCP = Host cell proteins

CPLL = Combinatorial peptide ligand libraries

RPC = Reversed-phase chromatography


HIC = Hydrophobic interaction chromatography

mAb = Monoclonal antibodies

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Received: July 10, 2010 Revised: November 10, 2010 Accepted: December 10, 2010

combinatorial chemistry

Documents

lowabundance proteins

rare proteins

thousands of proteins

rdna proteins

low sensitivity

host cell impurities

lowabundance proteome

selected host cells