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Amino Acid Analysis of Peptide Loading Ratios in Conjugate Vaccines: A Comparison of Direct Electrochemical Detection and 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Pre-Column Derivatization Methods Deborah D. Nahas,* Jennifer S. Palladino, Joseph G. Joyce, and Robert W. Hepler Merck Research Laboratories, 770 Sumneytown Pike, P.O. Box 4, WP16-107, West Point, Pennsylvania 19486. Received June 22, 2007; Revised Manuscript Received September 24, 2007

Amino acid analysis using direct electrochemical detection was compared with precolumn fluorescent derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for evaluation of the degree of covalent coupling of peptides to a carrier–protein complex derived from the bacteria Neisseria meningitidis. AQC derivatization was found to give superior sensitivity compared to electrochemical detection, with less interference from sample components such as carbohydrates or buffer salts. Hydrolysis time and temperature were optimized for maximal recoveries of the marker amino acid 6-aminohexanoic acid (ε-Ahx) and the unique amino acids S-dicarboxyethyl cysteine (SDCEC) and S-carboxymethyl homocysteine (SCHMC), which are generated upon the hydrolysis of the covalent linkage between the peptide and the carrier protein. Quantitation of these amino acids enabled the determination of the ratio of peptide to protein in the conjugate samples.

INTRODUCTION Certain classes of antigens, including peptides, carbohydrates, or polysaccharides, are often covalently coupled to carrier proteins in order to enhance their poor native immunogenicity. Such bioconjugates can be used as potential vaccine candidates or, in the cases of small peptides, as immunogens to generate antibodies against a particular target protein (1–3). The conjugation of poorly immunogenic molecules to carrier proteins, such as keyhole limpet hemocyanin (KLH), diphtheria toxoid, and Neisseria meningitidis outer membrane protein complex (OMPC), has been demonstrated to facilitate the induction of T-celldependent immune responses (4–6). OMPC is a multiprotein complex with associated lipids that has proven to be an excellent carrier in several licensed and experimental conjugate vaccines because of its unique immunostimulatory properties (7–9). Studies have shown that OMPC also possesses adjuvant-like properties based on its ability to activate B-cells through its lipopolysaccharide and protein components (4, 10). Additionally, neisserial porins are capable of activating B-cells by mediating toll-like receptor (TLR) signaling through the effector molecule MyD88 (11). Our group has prepared a number of covalent OMPC–peptide conjugates employing two different thiol-based coupling strategies. In the first strategy, OMPC was thiolated on surfaceexposed lysines with N-acetyl homocysteine thiolactone (NAHT) and then reacted with a bromoacetylated peptide. In the second strategy, lysine residues in OMPC were derivatized using the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) in order to introduce a maleimide group for reaction with a cysteinecontaining peptide. These thiol-based conjugation chemistries allow directional coupling due to the fact that the reactive thiol or haloacetyl functionality can be added at either the N-terminus or at the C-terminus of the peptide of interest. Another advantage is that the levels of activation can be monitored and controlled, * Corresponding author. Phone: 215-652-2198. Fax: 215-652-7320. E-mail: [email protected].

which is often critical to maintaining the solubility and stability of peptide–carrier conjugates. In the current study, the OMPC–peptide conjugates were analyzed by amino acid analysis, which is the technique most commonly employed to quantitate the molar ratio of peptide to protein in a conjugate (12, 13). This ratio is critical for calculating doses to be administered in immunization studies and assessing the comparability of different vaccine preparations. Amino acid analysis is also useful for monitoring the level of incorporation of the activating functionality on the protein carrier. In the case of sSMCC-mediated maleimidation, this is equivalent to the amount of tranexamic acid (Txa) generated upon acid hydrolysis of the conjugate. In this way, activation levels can be controlled, which is often critical to maintaining the solubility and stability of peptide–carrier conjugates. RP-HPLC with precolumn derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is compared with direct electrochemical detection of amino acids by high pH anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) for the analysis of OMPC–peptide conjugates. The gradient separations were optimized for both HPLC methods in order to resolve all 17 common amino acids: the peptide-derived spacer amino acid, 6-aminohexanoic acid (ε-Ahx); the activation reporter residue, tranexamic acid (Txa); and the coupling chemistry-specific amino acids S-dicarboxyethyl cysteine (SDCEC) and S-carboxymethyl homocysteine (SCMHC) generated by hydrolysis of the peptide–protein conjugate bond. The effects of hydrolysis time and temperature on the recovery of all relevant amino acids are also reported.

EXPERIMENTAL PROCEDURES Preparation of Peptide–OMPC Conjugates. Synthetic peptides were prepared by standard t-Boc or Fmoc solid phase synthesis. The spacer ε-Ahx was incorporated in the peptide between the primary sequence and the amino terminal cysteine or between the primary sequence and bromoacetyl group on the carboxy terminus. The thiol-based conjugation chemistries

10.1021/bc700232z CCC: $40.75  2008 American Chemical Society Published on Web 12/12/2007

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Scheme 1a

a A carrier protein is maleimide-activated with sSMCC, which reacts with primary amines (Lys residues and N-terminus). Then a cysteinecontaining peptide reacts with the maleimide-activated carrier protein to form a peptide–protein conjugate. Acid hydrolysis of the resulting conjugate liberates SDCEC and tranexamic acid in addition to the constituent amino acids of the peptide and the carrier protein. Quantitation of SDCEC by amino acid analysis can be used to determine the ratio of peptide to carrier protein.

used to couple the synthetic peptides to OMPC have been previously described in detail. Cysteinylated peptide Ac-EKTNEKFHQIEKEFSEVEGRIQDLEKYVENTKINLWSYNAEL-AhxC-NH2 was conjugated to OMPC that had been maleimidated with sSMCC according to the method of Fan, et al. (1), and the peptide bromoacetyl-Ahx-(Peg)AEDVGSNK-(Peg)-DAEFRHDSNH2 was conjugated to OMPC that had been thiolated using NAHT (14). For each conjugation protocol, a sample of activated OMPC without added peptide was carried through the reaction and desalting steps as a control. Amino Acid Analysis. Vapor phase hydrolysis of the OMPC peptide conjugates was performed with constant-boiling 6N HCl, 1% phenol at 110 °C for 20–70 h using an ELDEX (Napa, CA) H/D workstation. Protein hydrolysates and amino acid standards were derivatized with AQC using the Waters AccQ Tag Chemistry Package according to the manufacturer’s directions (15). AQC reacts with primary and secondary amines to form stable, fluorescent derivatives that can be separated by RP-HPLC (16–19). The fluorescent AQC derivatives were separated on an AccQ Tag Nova-Pak C18, 4 µM, 3.9 × 150 mm column using a modified gradient program (Modified Chromatography for Cell Culture Analysis; http://www.waters.com). Alternatively, protein hydrolysates and amino acid standards were dissolved in high purity deionized water and directly injected onto an AminoPac PA10 2 × 250 mm analytical column with a 2 × 50 mm AminoPac PA10 guard column (20, 21). Calculation of Peptide to OMPC Ratio. The amino acid composition of OMPC was empirically determined as the micromoles of each amino acid detected in a known amount of OMPC total protein (as determined independently by modified Lowry protein assay (23)). The following 11 hydrolysis-stable residues were used for routine quantitation: Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, and Val. The amount of OMPC in the OMPC–peptide conjugate sample was calculated from the empirically determined amino acid composition using those acidstable residues not present within the sequence of the peptide. The two thiol-based conjugation chemistries employed in this study result in conjugates that liberate unique, non-natural amino acids upon acid hydrolysis, in addition to the constituent amino acids of the peptide and the carrier protein (23). Conjugates prepared with a cysteinylated peptide and maleimide activated OMPC liberate SDCEC and tranexamic acid (Scheme 1), and conjugates prepared from NAHT-activated OMPC and a bro-

moactelyated peptide liberate SCMHC (Scheme 2) (14). Quantitation of these unique residues by amino acid analysis was used to determine the ratio of peptide to carrier protein. Also, since each mole of peptide contained a fixed and known amount of ε-Ahx (typically 1 or 2 mol ε-Ahx/ mol peptide), the molar amount of peptide in the conjugate was equivalent to the molar concentration of ε-Ahx, assuming complete removal of free peptide. Finally, the molar ratio of peptide to protein was calculated by quantitative comparison of conjugate and OMPConly compositions using the modified least-squares algorithm described by Shuler, et al. (12).

RESULTS The modified elution conditions developed for amino acid analysis of cell culture supernatants by RP-HPLC proved suitable for resolving the unique conjugate-specific amino acids SDCEC, SCMHC, ε-Ahx and Txa from the 17 common peptideand protein-derived amino acids within a single 60 min run. RP-HPLC analysis of the AQC derivatives provided a linear response for all components between 1.5 pmol and 50 pmol injected on the column. AAA Direct amino acid analysis by HPAE-PAD did not prove useful for the analysis of OMPC–peptide conjugates because of peaks that interfered with the detection of Arg and His, and the fact that HEPES buffer, which was used for conjugate preparation, interfered with the detection of leucine and caused excessive fouling of the gold electrode surface. Also, the method was considerably less sensitive than fluorescent detection of AQC-derivatized amino acids. The limit of detection for all amino acids using the AAA Direct method was 50 pmol, compared with 1.5 pmol for the AccQTag method. Comparison of Amino Acid Analysis by Direct Detection versus Precolumn Derivatization. The amino acid composition of a single lot of OMPC was determined following acid hydrolysis by an independent contract laboratory, using cation exchange chromatography with postcolumn ninhydrin derivatization (AAASL, Boring, OR). The results were compared with those obtained from the RP-HPLC method with precolumn AQC derivatization and direct analysis of hydrolyzed samples by HPAE-PAD (Table 1). In general, the differences between our AccQTag results and those of the contract laboratory were within experimental error (3–12% RSD). However, significant differences were observed between the

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Scheme 2a

a A carrier protein, such as OMPC, is activated with N-acetylhomocysteine thiolactone \(NAHT\), which reacts with primary amines \(Lys residues and N-terminus\). Then a bromoacetylated peptide can react with the NAHT-activated carrier protein to form a peptide–protein conjugate. Acid hydrolysis of the resulting conjugate liberates SCMHC in addition to the constituent amino acids of the peptide and the carrier protein. Quantitation of SCMHC by amino acid analysis can be used to determine the ratio of peptide to carrier protein.

Table 1. Amino Acid Composition of OMPC from Neisseria meningitidis contract laboratoryb amino acida arginine lysine alanine glycine valine isoleucine leucine histidine phenylalanine glutamate aspartate

average 0.3547 0.5211 0.8405 0.6979 0.6104 0.2846 0.4274 0.1454 0.2675 0.6493 0.6878

standard deviation 0.0130 0.0197 0.0502 0.0297 0.0231 0.0121 0.0198 0.0054 0.0112 0.0623 0.0279

AAA direct (HPAE-PAD)c % CV 4% 4% 6% 4% 4% 4% 5% 4% 4% 10% 4%

average d

0.7503 0.5523 0.7346 0.6195 0.5373 0.1999 0.3699 0.1291 0.2431 0.6948 0.7020

AccQTag(RP-HPLC FLD)c

standard deviation

% CV

average

standard deviation

% CV

0.0465 0.0489 0.0318 0.0201 0.0073 0.0244 0.0160 0.0216 0.0204 0.0725 0.0740

6% 9% 4% 3% 1% 12% 4% 17% 8% 10% 11%

0.3550 0.5029 0.7805 0.7307 0.5733 0.2300 0.4173 0.1212 0.2470 0.7178 0.7688

0.0084 0.0195 0.0332 0.0112 0.0244 0.0088 0.0147 0.0056 0.0103 0.0248 0.0281

2% 4% 4% 2% 4% 4% 4% 5% 4% 3% 4%

a Quantitation of acid-stable residues reported as the average µmol residue/mg OMPC ( standard deviation. b Nine replicates were analyzed in a single run, using postcolumn ninhydrin detection. c Three replicates were analyzed on three separate days. d An unresolved peak that is also present in the buffer blank interferes with the detection of arginine.

AAA Direct method and the other methods. The AAA Direct method resulted in significantly lower values for alanine, glycine, valine, isoleucine, and leucine, and 2-fold higher concentrations for arginine (because of an interfering peak). The average variability observed with the AAA Direct method (8% RSD) was also twice as high as the average variability of the AccQTag method (4% RSD). Optimization of Hydrolysis Conditions for Peptide/ Carrier Conjugates. Because the AAA Direct analysis was less sensitive and was affected by interferences from buffer components, we focused hydrolysis optimization efforts solely on the AccQTag method. Hydrolysis time and temperature were evaluated in order to maximize the recoveries of the conjugatespecific unique amino acids. Acid hydrolysis of amino acid standards with 6 N HCl and 1% phenol revealed a 24% reduction in peak area for SCMHC and a 13% reduction in peak area for SDCEHC within 20 h at 110 °C. However, peak areas for SCMC, SDCEC, ε-Ahx, and Txa remain stable even after 70 h of hydrolysis. Peptide–carrier conjugates prepared from both thiolated and maleimide-activated OMPC were also analyzed with hydrolysis times ranging from 20 to 70 h at 110 °C. The average results for the ε-Ahx/OMPC ratio, SDCEC/OMPC ratio, and SCMHC/ OMPC ratio were then calculated for the various different hydrolysis conditions, along with the peptide loading determined by the LSA spreadsheet method. For both conjugates, the total protein concentrations did not vary significantly with increasing

Figure 1. A conjugate prepared from a bromoacetylated peptide and thiolated OMPC was hydrolyzed at 110 °C for 17.5–70 h. The hydrolyzates were derivatized with AQC and analyzed by RP-HPLC. The ratio of peptide to OMPC was then calculated from either (a) the amount of ε-Ahx linker, (b) the amount of SCMHC, or (c) using the least-squares regression method of Shuler, et al.

hydrolysis time (data not shown). The ratio of peptide to OMPC calculated from the concentration of SCMHC was essentially constant with hydrolysis time, as was the ratio of ε-Ahx to OMPC (Figure 1), even though a standard of SCMHC was shown to degrade at hydrolysis times longer than 20 h. Interestingly, for the conjugate sample that was prepared from

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ACKNOWLEDGMENT We thank Jay Gambee at AAA Service Laboratory, Inc. for performing amino acid analysis, Chengwei Wu for peptide synthesis, and Dr. Craig Przysiecki for providing unique amino acid standards. Supporting Information Available: Hydrolysis time course of amino acid standards, RP-HPLC chromatogram of amino acid standards, and HPAE-PAD chromatogram of amino acid standards. This information is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED Figure 2. A conjugate prepared from a cysteine containing peptide and maleimide-activated OMPC was hydrolyzed at 110 °C for between 17.5 and 70 h. The hydrolyzates were derivatized with AQC and analyzed by RP-HPLC. The ratio of peptide to OMPC was then calculated from either the amount of ε-Ahx linker or the amount of SDCEC.

maleimide-activated OMPC, 70 h of hydrolysis at 110 °C was required to obtain maximal recovery of SDCEC (Figure 2). The need for extended hydrolysis times in order to quantitatively recover SDCEC from thiol–maleimide adducts has been previously demonstrated (23).

DISCUSSION Amino acid analysis provided a reliable means for assessing the covalent attachment of peptide to carrier protein because acid hydrolysis of the nascent conjugate bonds generates novel amino acids such as SDCEC or SCMHC, depending on the choice of chemistry used. The total amount of peptide in the complex can also be quantified by monitoring the concentration of a unique peptide-specific component, such as the ε-Ahx linker described here. In the current report, ratios of peptide to OMPC carrier protein were calculated using both methods, with equivalent results. Of the methods for calculating the peptide content of the conjugate, only measurement of the unique amino acid formed upon hydrolysis of the conjugate bond provides conclusive evidence of the covalent attachment of the peptide to the protein. Incomplete removal of aggregated peptide from OMPC conjugate samples during dialysis can lead to overestimation of the amount of peptide incorporated into the carrier when this ratio is calculated using either the LSA approach or the quantitation of ε-Ahx. Importantly, our results clearly demonstrate that optimization of the hydrolysis conditions used for amino acid analysis is critical to obtaining consistent and accurate results when utilizing unique amino acid residues. In our hands, ε-Ahx was quantitatively recovered following the hydrolysis of peptide–OMPC conjugates under relatively standard conditions (15–20 h), whereas a more extensive hydrolysis time of 70 h was required for SDCEC. In contrast, SCMHC was recovered quantitatively in hydrolysates prepared at 20 h, and the ratio of SCMHC to OMPC did not change significantly with longer hydrolysis times. Our observations, to a large degree, are likely related to the complex nature of a multicomponent lipoprotein carrier such as OMPC, and the results of a similar comparison may be quite different for peptides coupled to a soluble monomeric protein carrier such as bovine serum albumin, even when employing the same conjugation chemistries. These differences exemplify the requirement for rigorous optimization of experimental parameters when seeking to employ these quantitative methods in a reliable and reproducible manner.

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