Approaches to Analyzing Therapeutic Peptides and Proteins by LC-MS/MSMatthew Ewles, Covance Laboratories Ltd., Harrogate, UK and Mary Pelzer, Covance Laboratories Inc., Madison, WisconsinCorresponding author: matthew.ewles@covance.com

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Introduction Demand for analysis of therapeutic peptides and proteins for toxicokinetic and pharmacokinetic studies is increasing as more protein and peptide drugs enter clinical development. More than 600 biological drugs are currently approved by the U.S. Food and Drug Administration, and biologicals (including peptides, proteins and other novel therapeutics such as nucleotides) now account for 30% of all drugs in development and a more than half of new molecular entities.For purposes of bioanalytical method development we distinguish peptides from proteins based on size: Peptides contain approximately fifty or fewer amino acid residues, with molecular weights of about 6,000 Da or smaller; proteins comprise everything above this cutoff. Many of the potential therapeutically significant biomolecules are 4,000 Da or smaller and are therefore classified as peptides.Our interest in analyzing peptides and proteins arises from the need, during preclinical and clinical studies, to quantify levels of protein and peptide drugs and their metabolites (the process of bioanalysis). To support the predicted increase in peptides and proteins undergoing such studies, we expect that the demand for immunological and LC-MS/MS bioanalytical methods to increase. We have been working for the last several years to develop bioanalytical methods for accurate, selective and sensitive quantification of peptide and protein

therapeutics. Developing and validating LC-MS/MS methods for proteins and peptides can be challenging, and presents additional difficulties over conventional methods for small molecule therapeutics. This article draws on our personal experience and knowledge to discuss some of these difficulties, and how they are overcome by the bioanalytical teams at Covance Laboratories.

Two Predominant TechniquesImmunoassays and LC-MS/MS have emerged as the two main approaches for quantifying peptides and proteins in biological samples. Immunoassay methods are highly sensitive, and are able to measure very low concentrations of larger molecules where MS/MS may not achieve adequate sensitivity. They also require minimal sample preparation and are available in convenient and high-throughput platforms. However, immunoassays often suffer from poor linear quantification ranges and require

antibodies to be raised specific to the target analyte, which can be expensive and time consuming. From the point of view of a bioanalytical method, the most serious drawback of immunoassay is the interference observed from homologous peptides and high-abundance proteins present in the sample. This adds to poor selectivity between structurally and

chemically similar peptides or proteins. For example, parent and metabolite compounds may differ by only a small chemical change outside of the region recognized by the antibody.

Advantages LC-MS/MS Offers Over Immunoassay

• Selectivity between structurally or chemically similar peptides and proteins

• No requirement for antibodies• Improved precision and accuracy• Higher throughput

Peptide and protein bioanalysis by LC-MS/MS can offer several advantages over immunoassay, including improved precision and accuracy, higher throughput, and most importantly, the orthogonal resolving power of LC and MS/MS. This allows for structurally or chemically similar peptides and proteins to be distinguished from each other, where poor selectivity would generate a false signal by immunoassay methods. LC-MS/MS in particular has significant advantages for quantification of peptides where highly selective antibodies cannot be raised, and LC-MS/MS can often be the only approach for achieving adequate selectivity and sensitivity. Additional challenges that occur when developing LC-MS/MS methods for peptides and proteins include:

The m/z of ionized molecules may exceed the �instrument’s capabilities.Multiple charging and wide isotopic distribution �limits achievable sensitivity when monitoring only a single charge state.Folding or conformational changes due to solvent �composition may affect the charge state and ionization of the molecule.Ionization may be concentration dependent, causing �nonlinear response.Poor fragmentation efficiency of molecules within �the collision cell.Many proteins and peptides display poor solubility in �organic solvents. An abundance of chemically similar endogenous �proteins and peptides in plasma and serum samples causing selectivity and recovery issues.Extensive metabolism and �modification of the target analyte.Adsorption of peptides and proteins �to various surfaces.Protein-protein interactions resulting in protein �binding and poor recovery.Chemical, enzymatic or proteolytic degradation of �the target molecule.Lack of suitable isotopically labeled internal �standards.

Notes on Sample and Analyte HandlingPeptides and proteins often demonstrate significant adsorption to vessel walls at low concentrations. It is therefore important to test various vessels for adsorption

at the start of a project. We use low-adsorption polypropylene and polyethylene vessels to minimize adsorption. Where these do not eliminate adsorption, we try silanized glass vessels, in which the silanol groups (which are often responsible for nonspecific adsorption) have been chemically inactivated. Adsorption from solutions prepared in plasma or serum is far less significant than matrix-free solutions due to blocking of

adsorption sites by endogenous proteins. To prevent adsorption, preparation of low concentration matrix-free solutions of any protein or peptide, particularly of predominantly aqueous composition, should be avoided. Protein or peptide

analytes should be spiked from a high concentration stock (where any adsorption losses are insignificant) directly into plasma or serum, and subsequently serial-diluted in the same plasma or serum to prepare calibration standard and quality control samples. Where matrix-free solutions are required (e.g. for reference standards, MS/MS test solutions or urine assays of some smaller peptides), the solubility may be enhanced (and adsorption inhibited) by using organic-aqueous mixtures or by adding surfactants or bovine serum albumin (BSA) to block adsorption sites. For sample extracts, organic solvent (usually acetonitrile) should be present at greater than 20% to limit adsorption. However, this can be modified to reduce a negative effect on chromatography.

Analyte StabilityBlood, plasma and serum samples contain proteases and other enzymes that modify the structure of proteins and

peptides. It is therefore important to ensure the stabilization of the analyte at the start of the project. We have determined that 20 mM serine protease inhibitor [diisopropylfluorophosphate (DFP)] performed better than many standard protease inhibitor cocktails

[sodium fluoride/potassium oxalate and trichloroacetic acid (TCA)] in stabilizing plasma solutions stored frozen for one month (Addison et al., 2010). Pefabloc® is another commonly employed stabilizer for peptides and proteins. It contains 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) that irreversibly inhibits serine proteases without the solubility and toxicity drawbacks of DFP or phenylmethylsulphonyl fluoride (PMSF). The choice of which stabilizer to use is compound dependant and often simply acidifying the sample with formic or hydrochloric acid is adequate to inhibit proteases and stabilize the compound. Storage of samples below -60°C

Adsorption may be inhibited using organic-aqueous mixtures, or by adding surfactants or bovine serum albumin.

The choice of which stabilizer to use is compound dependant, and often acidifying the sample is adequate to inhibit proteases.

and handling on ice wherever possible will slow down the rate of any biological reactions and help limit residual protease activity or further enzymic modification of the analytes.Our recent work has examined the applications of dried blood spotting (DBS) for analysis of therapeutic peptides. Blood may be spotted onto treated paper containing surfactants and enzyme inhibitors to denature proteins, and is allowed to dry. This prevents proteolytic analyte degradation. We have validated a method for a 2,554 Da peptide (ramoplanin) using this approach and demonstrated acceptable precision and accuracy, selectivity and analyte stability. Furthermore, this work demonstrates robust performance of the dried blood spotting technique for peptide therapeutics (Ewles et al., 2010).

Sample Cleanup from Endogenous ProteinsThe abundance of endogenous proteins in plasma and serum is shown in Figure 1. More than 90% of the endogenous protein is comprised of only a handful of different proteins. The peak around 65 kDa comprises mainly albumin, which at a concentration of 30-50 mg/mL accounts for up to 60% of total serum protein content.

The shoulder at 100 kDa comprises several immunoglobulins, each at concentrations of approximately 1 mg/mL accounting for 30-35% of total serum protein. These high endogenous protein concentrations pose a challenge to LC-MS/MS analysis. Achieving analyte separation prior to MS/MS is essential to avoid matrix effects or an abundance of interfering peaks caused by the

wide isotopic distribution of the large endogenous proteins and peptides.Endogenous proteins and peptides are difficult to remove from samples due to their chemical similarity to the target analytes. The twenty most abundant amino acids all possess the characteristic carboxylic acid and amine functional groups and differ only by the group on the alpha carbon. This group imparts the unique chemical characteristics to the amino acid. Most peptides and proteins contain a mixture of most chemical categories of amino acids, including:

hydrophilic (alanine, glycine, leucine) �basic (arginine, lysine, histidine) �acidic (aspartic acid, glutamic acid) �aromatic (tyrosine, phenylalanine) �

Therefore, finding selective chemical properties to exploit for achieving sample cleanup for a protein or peptide analyte can be challenging. When developing a method, it is important to look at the relative abundance of different chemical groups and the chemical behavior of the whole protein or peptide molecule. Molecules with a high abundance of polar amino acids may demonstrate poor solubility in organic solvents or aqueous-organic mixtures used during bioanalytical extraction and chromatography,

and may require additional precautions. Molecules containing a large number of arginine residues will have a strongly basic character, which can be exploited when developing extraction and chromatographic methods to achieve selectivity against other, less basic, endogenous proteins or peptides.Because endogenous proteins are usually much larger than therapeutic peptides, the most straightforward way to eliminate them is through protein precipitation. This technique involves mixing the plasma or serum sample with a water-miscible organic solvent (typically acetonitrile or methanol) at a ratio of at least two parts solvent

to one part sample. This precipitates larger proteins (including most of the endogenous proteins), which can be removed from the sample by centrifugation. Proteins precipitate at varying concentrations of organic solvent. Larger proteins typically precipitate at >40% solvent, whereas smaller proteins may require a greater proportion

Figure 1. The relative abundance of amino acids, peptides and proteins in human serum on the basis of molecular weight.

of solvent to precipitate. Peptides generally do not precipitate at all, although if they are particularly polar, their solubility may be compromised by this high-organic solvent approach. Furthermore, we observe a tendency of some peptides to bind to the precipitated protein pellet, leading to poor and often variable recovery. In these cases, aqueous acid precipitations can be employed instead, allowing precipitation with up to 10% trifluoroacetic acid (TFA) or TCA in an organic composition suitable for analyte solubility. Such high concentrations of acid must be removed from the sample before it can be injected onto the LC-MS/MS [either through evaporation or solid-phase extraction (SPE)] to avoid interferences with the chromatography.Protein precipitation provides a quick and simple technique for peptide analysis, and when coupled to high-resolution LC-MS/MS it can often yield very good results. Unfortunately, protein precipitation provides a very nonselective cleanup that removes only larger protein interferences and it cannot be further optimized for additional selectivity based on the chemistry of the analyte. Failure to remove enough endogenous material during sample cleanup can result in matrix effects (ion suppression, enhancement or protein binding) or endogenous peaks, and therefore if these effects are observed during method development, a more rigorous and analyte-specific sample cleanup may be required.Where additional cleanup and selectivity is required, SPE may be used either instead of (or in addition to) protein precipitation. SPE offers several advantages over protein precipitation:

Avoids subjecting the analyte to harsh organic �conditions that can compromise the solubility of more polar peptides or proteins.

Any analyte demonstrating poor solubility by -protein precipitation may yield better results by SPE.

Allows for larger sample volumes to be extracted and �loaded onto the sorbent, thus allowing lower limits of quantification to be achieved. Can allow the elimination of protein binding by �incubating the sample with urea, guanidine or strong acid prior to loading.

The peptide is maintained in an aqueous -environment which is more conducive to their solubility. The salt disrupts protein-protein binding prior -to loading but is eliminated (along with the endogenous proteins) during loading and washing.

Can be optimized based on your analyte chemistry �to obtain better sample cleanup than protein precipitation. SPE chemistries based on C18-type nonpolar interactions tend to provide only limited retention to polar biological compounds.

If the SPE is being used only for the purpose of -sample desalting or solvent exchange following an aqueous acid precipitation or protein denaturation, only weak retention is required.

Anion or cation exchange SPE is usually more useful. These allow loading and elution under primarily aqueous

conditions, utilizing pH switching to control retention and elution of your analyte. Using ion exchange SPE for sample cleanup followed by reverse phase chromatography provides a two-dimensional separation prior to MS/MS, which can help avoid matrix

effects or interferences from endogenous molecules. For the purposes of eliminating larger proteins from a sample, SPE can also take advantage of size-exclusion principles. Depending on the size of the therapeutic peptide or protein, one can select an SPE sorbent with a pore size appropriate to the separation. For example, a sorbent with pore sizes of 40-80Å will retain any protein smaller than roughly 20 kDa, effectively eliminating larger and higher-abundance interfering proteins by size

exclusion. Samples may then be cleaned up further through reverse phase or ion exchange approaches.Unfortunately, SPE methods require more work to develop and optimize and, as they utilize specific chemical interactions, without an isotopically

labeled internal standard (which mimics the chemical interactions of the analyte) they have an increased risk of showing discrepant recovery between the internal standard and analyte. Conversely, SPE methods generally produce cleaner extracts, making matrix effects on both the analyte and internal standard less likely.

Analyte Heterogeneity Analysis of proteins by LC-MS/MS is further complicated by the heterogeneity of therapeutic proteins. Mammalian protein expression systems result in several isoforms of some proteins being produced due to post-translational modifications, including:

SPE can be adapted to overcome issues of poor selectivity, sensitivity, insolubility and protein binding.

Ion exchange SPE followed by reverse phase chromatography provides two-dimensional separation for maximum selectivity.

amidation �glycosylation �acetylation �methylation �

disulphide bond formation �hydroxylation �oxidation �misfolding �

Some of these modifications affect the observed target molecular weight, and MS/MS analysis of proteins will sometimes show a range of molecular ion peaks. More significant modifications (particularly glycosylation) can have a substantial effect on the molecular weight, with the impact on small peptides being far more pronounced than on larger proteins (as a percentage to the total molecular weight).

Tryptic DigestionThe use of proteases to perform controlled digestion of larger proteins to produce smaller surrogate peptides can help overcome some of the MS/MS challenges. Using tryptic digestion for protein therapeutics above 10 kDa can provide a method that competes with immunoassay in terms of sensitivity, but with the specificity provided by MS/MS.One drawback of tryptic digestion is that it risks compromising the selectivity of the method, due to monitoring only one surrogate peptide that might not fully represent the entire molecule. If any metabolism or modification of the protein is suspected, additional surrogate peptides covering the suspected sites at which the metabolism or modification is occurring can be monitored. This allows various isoforms to be distinguished and metabolites to be separately quantified. For proteins that are extensively modified, more surrogate peptides are monitored, increasing the coverage of the intact molecule in terms of selectivity. Immunoassay does not allow for targeted selectivity toward sites of metabolism or modification and does not provide the required level of selectivity for many assays.Analyzing proteins by tryptic digestion is more complicated than peptide analysis and introduces several unique challenges for method development. Each protein and sample requires individualized optimization of digestion and cleanup, since endogenous protein contaminants will also be digested and interfere with analysis. We witnessed that if plasma sample digests are not adequately cleaned up prior to MS/MS a huge number of endogenous peaks are observed for the tryptic peptides of endogenous plasma proteins with an m/z overlapping that of the chosen surrogate peptide. We would generally expect to require off-line and on-line SPE to achieve adequate cleanup of the surrogate peptides from a crude plasma or serum digest, and even then, we anticipate a large number of endogenous peaks that will require good chromatographic separation from our surrogate peptide of interest.

To avoid the issues with isobaric peptides produced from digestion of abundant plasma proteins interfering with the performance of the assay, it is desirable for smaller analytes (<20 kDa) to be separated from the much larger abundant proteins (e.g. albumin and immunoglobulins) prior to digestion. To achieve this, one can take advantage of SPE size-exclusion principles to prevent the retention of large abundant proteins or partial protein precipitation to eliminate larger proteins. Various commercially available kits allow for immunoaffinity removal of the abundant proteins. With these depleted, digestion becomes more efficient, and numerous endogenous interferences are almost completely eliminated.To improve the throughput of tryptic digestion methods by avoiding the overnight incubation and digestion with enzymes, we often use organic solvent to accelerate the reaction. We found that 50% methanol accelerates the enzymatic reaction in complex systems such as plasma. Methanol denatures the target protein, making it more accessible to the trypsin, allowing the reaction to reach

completion much quicker and more efficiently. Li found that this reduced digestion time to about 30 minutes and that the technique was qualitatively and quantitatively equivalent to overnight digestion (Li et al., 2009). As an internal standard Li used a second

protein, digested along with the target analyte to compensate for possible variability in the efficiency of the digestion reaction. Locating suitable internal standards for use with a tryptic digestion approach is problematic. One must first decide, and sometimes compromise, on which steps of the extraction are to be internally standardized. Ideally internal standardization should start from when abundant proteins are depleted from the test article (or at the digestion if depletion is not being used). The internal standard protein should therefore be of similar size and polarity to the test compound. It should digest to produce a surrogate peptide of similar size and polarity to the surrogate peptide of the analyte. A homologous protein or stable-labeled protein is ideal, but these are expensive to manufacture and are rarely available. One must sometimes compromise and it might be useful to have a catalog of candidate internal standard proteins available over a range of molecular weights.

HPLC AnalysisAfter sample preparation we perform LC-MS/MS analysis with reverse phase HPLC, occasionally HILIC for smaller peptides or UHPLC. UHPLC employs 1.7 μm particles and higher pressures for improved

To eliminate large and abundant endogenous protein interferences, SPE size-exclusion principles can be exploited.

chromatographic resolution, and allows for shorter runtimes. Reverse phase chromatography allows separation of structurally and chemically similar peptides and the solvents are compatible with electrospray ionization.Reverse phase retention mechanisms for peptides and proteins differ from that of small molecules. For small molecules, partitioning occurs between the mobile phase and stationary phase as the analyte travels along the column. Very small peptides (<3,000 Da) are retained by similar mechanisms and are able to penetrate the pores of conventional reverse phase media, allowing for full interaction with the surface chemistries. However, larger peptides and proteins interact only with the surface and adsorb without fully penetrating the pores within the particles. They are desorbed at a critical organic solvent concentration, unique to each peptide or protein. Larger-pore stationary phases (around 300Å) do allow penetration of larger molecules into the particle and may provide much greater selectivity and separation for larger peptides and proteins.Where the adsorption-desorption mechanism dominates, factors such as column length have minimal impact on the chromatography. Isocratic chromatography generally produces poor results due to adsorption-desorption and therefore gradient chromatography is preferred to achieve optimum peak shape. However, other factors such as mobile phase composition and gradient rate play as much of a role in controlling retention as they do for small molecules.Organic modifiers are almost always employed in protein and peptide analysis. Typically acidic modifiers are used as they neutralize the weakly acidic carboxy groups, resulting in less net charge on the molecule and better reverse phase retention and chromatography. Charged molecules often exhibit poor retention or secondary interactions by reverse phase chromatography, resulting in very poor peak shape and separation. Formic or acetic acid is suitable for many peptides; however, for larger or strongly basic peptides or proteins, where poor retention or poor chromatography is observed, TFA is commonly used. TFA can enhance reverse phase chromatography methods by ion-pairing with basic groups thus reducing the overall charge on the molecule and enhancing its retention. Unfortunately, TFA strongly suppresses mass spectrometer sensitivity and therefore a balance must be struck between chromatographic selectivity and MS/MS sensitivity. We

commonly use TFA concentrations between 0.01% and 0.5% in mobile phases. The concentration depends on whether that particular analyte would benefit more from:

higher chromatographic retention �better peak shape or greater selectivity �

employ higher TFA concentrations for peptides -and proteins that are poorly retained due to an abundance of basic groups such as lysine and arginine

greater MS/MS sensitivity �employ lower TFA concentrations or use formic -acid instead

Additionally, the use of columns such as the C-18 peptide column or the C-18 SymmetryShield™ column from Waters (Milford, Massachusetts, USA) can help overcome some reverse phase chromatography issues. SymmetryShield™ contains a polar embedded phase that

shields the residual silanol groups on the surface of the bonded phase from interacting with highly basic analytes (for example peptides and proteins rich in lysine residues), thereby eliminating their detrimental influence on the separation. If good peak shape or

separation by reverse phase approaches can not be obtained for very acidic or basic proteins or peptides, ion exchange chromatography may also be worth investigating.

MS/MS ChallengesThe maximum m/z of the triple quadrupole instruments used for bioanalysis is typically limited to 2,500-3,000. It is therefore advantageous that peptides greater than about 2,500 Da tend to multiply charge under electrospray conditions, thus reducing their m/z into the analytical range. Most peptides below 4,000 Da predominantly form the m+2 charge state under electrospray conditions.

Larger peptides and proteins tend to form additional m+3 and m+4 charge states, resulting in the signal being distributed between several different ions. Some peptide’s ratio of formation of one charge state to another can vary depending on solvent composition,

MS/MS parameters, or worse (from the point of view of achieving a linear response) with the concentration of the analyte and other molecules in the ionspray droplets. Analysis of proteins and peptides by mass spectrometry is further complicated by the natural abundance of 13C isotopes (around 1%). Larger molecules have a greater likelihood of possessing at least one or more 13C isotopes

Monitoring of several predominant charge states or isotopes and summing the data can sometimes gain additional sensitivity and balance concentration-dependant ionization effects.

TFA can enhance reverse phase chromatography by ion-pairing with basic groups thus improving the retention and peak shape.

and consequently demonstrate a wider isotopic ion distribution, thus limiting the abundance of any one m/z value. For both of these issues, monitoring of several predominant charge states or isotopes and summing the data can sometimes gain additional sensitivity and balance concentration-dependant ionization effects. However, this has the same effect as lowering the MS resolution and greatly increases the likelihood of encountering nonspecific peaks and matrix effects, as well as higher background noise. For larger proteins, digestion to produce smaller surrogate peptides can help overcome these issues, allowing a much greater MS/MS response to be achieved compared to the parent protein.Peptides and proteins tend to exhibit poor fragmentation efficiency within the collision cell of triple quadrupole instruments. This was demonstrated during our work with the therapeutic peptide ramoplanin (molecular weight 2,554 Da) (Ewles et al., 2010). Ramoplanin exhibits limited fragmentation under conditions that would usually and extensively fragment small (nonpeptide) molecules. Two selective fragments are produced at higher collision energy, but the fragmentation is incomplete and the majority of parent ions remain unfragmented, leaving a low abundance of these selective fragments. Increasing the collision energy further causes complete fragmentation of the molecules to small fragments of individual amino acids, which do not provide adequate selectivity toward any one protein or peptide analyte over endogenous proteins and peptides. Therefore, sensitivity is often limited by how readily your analyte fragments in the collision cell.

Three Approaches to Peptide and Protein AnalysisOwing to the difficulties discussed, we have developed a number of strategies to achieve robust, accurate and precise methods for protein and peptide LC-MS/MS quantification. We have adopted three broad approaches to act as a starting point for method development to achieve adequate sample cleanup and MS/MS performance, depending on the molecular weight of the analyte (Figure 2).

Approach One for Small PeptidesPeptides smaller than approximately 1.5 kDa tend to behave like small molecules in terms of their solubility, extractability and LC-MS/MS behavior, although they still demonstrate a tendency for adsorption and sometimes protein binding. We generally employ methods that are similar to those used for small molecule analysis. The peptide is handled to minimize adsorption and protein precipitation is initially assessed using methanol or acetonitrile. If this yields poor selectivity or the solubility or recovery of the peptide appears to be compromised, off-line SPE using reverse phase or ion exchange will be assessed. If necessary, extracts will be concentrated using heated nitrogen evaporation, to achieve the required sensitivity or to prevent solvent effects on the chromatography.

LC-MS/MS analysis is then performed using reverse phase chromatography (or HILIC as some small peptides demonstrate adequate solubility in acetonitrile) using HPLC or UHPLC. If the peptide is rich in arginine or lysine residues, the chromatography may need to be further developed to combat any problems caused by poor retention or poor peak shape.This approach allows analysis of many small peptides at plasma concentrations in pg/mL levels. Figure 3 shows a chromatogram for vasopressin, extracted from human plasma spiked at a concentration of 100 pg/mL using approach one with mixed-mode ion exchange and HILIC

Figure 2. Overview of our three approaches for protein and peptide quantification.

UHPLC. Achieving much lower limits of quantification should be possible with further work (Vincent et al., 2009).

Approach Two for Mid-Mass

Peptides and Small ProteinsOur second approach is used for analyzing peptides in the middle mass range between 1.5 and 10 kDa. Mid-mass peptides tend to demonstrate poor solubility in organic solvents, making protein precipitation and HILIC impossible. They also demonstrate more variable chemistry, which can interfere with reverse phase retention and separation. Molecules of this size can also be immunogenic and are often predominantly antibody-bound in plasma or serum. These size molecules may still be analyzed intact by MS/MS due to multiple charging reducing the m/z, although the achievable sensitivity is therefore compromised.Our second approach is to perform off-line SPE using mixed-mode ion exchange with sample loading in high concentrations of urea or guanidine to eliminate suspected protein binding. In some circumstances where large volumes of plasma are processed, it is often useful to perform protein precipitation on the samples with 10% TFA and then SPE on the supernatant. Not only does TFA abolish protein-protein interactions, but the precipitation eliminates larger proteins, thus preventing overloading and blocking of the sorbent. This is often seen when sensitivity requirements force sample volumes greater than 200 μL to be taken for analysis. After SPE, the extract is diluted with water containing small amounts of surfactant. This adjusts the polarity to more aqueous conditions and minimizes adsorption from these final extracts. At this point the entire sample is subjected to a second SPE using the Symbiosis on-line SPE instrument (Spark Holland, Emmen, Netherlands).The additional cleanup concentrates the sample prior to eluting onto the LC column, thereby maximizing sample loading for additional sensitivity. The on-line SPE is typically reverse phase (C18), which provides orthogonal separation in combination with the off-line ion exchange extraction. The on-line extraction can be optimized to separate or eliminate interferences and ensure that the additional loading does not result in gains to the background noise or interferences. Where very low limits of quantification are not required, the need for the on-line SPE step may be assessed. Reverse phase chromatography

using mobile phases containing TFA usually produces the best peak shape and separation for these mid-sized molecules.This approach has allowed us to achieve quantification of mid-mass peptides at concentrations as low as 10 pg/mL in plasma or serum. However, this depends on the sample volume, extraction recovery and efficiency of ionization and fragmentation.

Approach Three for ProteinsOur method for proteins above 10 kDa involves using trypsin to digest the protein at fixed positions (arginine and lysine resides) into smaller surrogate peptides and analyze these surrogate peptides, as described for approach two. For smaller molecules (<20 kDa) larger abundant proteins are first eliminated by size-exclusion using a simple reverse phase SPE. Elution with a solvent containing methanol can allow subsequent accelerated digestion. Alternatively, protein precipitation using a controlled ratio of organic solvent can be used to precipitate larger abundant proteins without affecting your analyte. Larger proteins (>20 kDa) are more difficult to separate from abundant plasma proteins. Where quantification of these is required, either the plasma will be directly digested or immunoaffinity purification may be investigated. Proteins are denatured with high concentrations of salt (such as urea or guanidine) and a reducing compound (such as beta-mercaptoethanol or dithiothreitol) to reduce disulphide bridges. This unravels the secondary and tertiary structures, allowing for overnight digestion with trypsin (or accelerated digestion in the presence of methanol). The surrogate peptides are then cleaned up and concentrated using off-line SPE followed by on-line

Figure 3. Vasopressin in human plasma analyzed using approach one to achieve 100 pg/mL lower limit of quantification using stable label (d5) internal standard.

SPE and LC-MS/MS similar to that adopted for approach two to achieve maximum loading and selectivity. We have previously reported the feasibility of approach three for quantification of Lysozyme (14.3 kDa) in human plasma over the range 50-50,000 ng/mL. The method involves depletion of abundant endogenous proteins using SPE, tryptic digestion, off-line and on-line SPE and LC-MS/MS analysis of a surrogate peptide without internal standardization (this work remains ongoing) (Ewles et al., 2010). Figure 4 shows a chromatogram of an extract of lysozyme at the lower limit of quantification of 50 ng/mL in plasma (3.5 nmol/L). We have also assessed the feasibility of this approach for quantification of a larger (52 kDa) protein using an analog protein as an internal standard. Separation from endogenous plasma proteins by size was not possible due to the high mass of the test article and so the plasma was digested without any pre-cleanup or depletion of abundant proteins. A chromatogram showing detection at 1,000 ng/mL in plasma (20 nmol/L) is included in Figure 5. This method was developed without on-line SPE and therefore we expect that lower limits of quantification should be achievable through implementation of this technology.

Figure 5. Chromatogram showing a 52 kDa protein analyzed using approach three at a concentration of approximately 1,000 ng/mL in human plasma. The analyte and internal standard peaks are indicated.

Figure 4. Chromatograms showing detection of Lysozyme in human plasma using approach three in blank plasma and plasma spiked at 50 ng/mL.

SummaryTherapeutic proteins and peptides in complex biological matrices such as plasma and serum present numerous challenges for bioanalytical method development, such as:

high concentrations of endogenous proteins �binding of targets to high-abundance proteins and �antibodiessolubility issues �nonspecific adsorption effects �instability �poor MS/MS behavior �chromatographic behavior �

These issues manifest themselves differently from one peptide to another and must therefore be addressed by the method development scientist on an analyte-by-analyte basis. However, it is useful to have some general guidance to assist with method development, and from our experience we have developed three standard approaches for achieving a robust LC-MS/MS method. These provide a good starting point, and have been extensively applied to the development and validation of many successful and robust peptide bioanalytical assays at Covance, often achieving quantification at low pg/mL concentrations.For more information on how Covance’s global bioanalytical services can support your drug development efforts, please call us at +1.888.COVANCE or +44.1423.500888.

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ReferencesAddison TE, King MJ and Larson MJ. Investigation of conditions necessary to stabilize a polypeptide therapeutic in plasma to support early drug development and analysis by LC/MS/MS. Poster presented at the 8th Annual Land O’Lakes Bioanalytical Conference, Merrimac, Wisconsin, 9–13 July 2007.

Ewles MF, Turpin PE, Goodwin L and Bakes D. Validation of a bioanalytical method for the quantification of a peptide therapeutic in human dried blood spots using LC-MS/MS. 2010; submitted for publication.

Ewles MF, Goodwin L and Bakes D. Feasibility assessment of a bioanalytical method for quantification of a 14.3 kDa protein in human plasma using tryptic digestion LC-MS/MS without a requirement for antibodies. Chromatography Today 2010; 3(1): 26-29.

Li F, Schmerberg CM and Ji QC. Accelerated tryptic digestion of proteins in plasma for absolute quantitation using a protein internal standard by liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2009; 23(5): 729-732.

Vincent J, Thomas-Oates J and Turpin P. Method development, optimisation and validation of a turbo ion spray LC-MS/MS bioanalytical assay for the quantitative analysis of arginine-8-vasopressin (AVP) in human plasma. A poster presented at the Analytical Research Forum 2009.