Abstract

AbstractA semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins.Keywords: Amino acid analysis, OPA-derivatization, reverse-phase HPLC, validation Amino-acid analysis has a long history in the characterization of protein-based products, since it provides information on the product concentration without referring to an external protein standard and it is independent from the shape and the charge of the protein. In addition, the determined amino-acid composition can confirm sample identity and gives a measure of sample purity. Furthermore, when combined with absorbance measurements, it allows the determination of extinction coefficients under various conditions.1 For protein conjugates, where the synthetic counterpart modifies the protein absorption properties, amino-acid analysis may be required as the only reliable quantification method.However, in spite of these features, few laboratories can perform such analysis in a reliable and quantitative way, due to the need for specialized equipment and skills. Usually, techniques based on ion-exchange separation coupled with post-column derivatization (e.g., with nin-hydrin, the “classical” method) are considered more precise1 than those based on pre-column derivatization and reversed-phase high-performance liquid chromatography (RP-HPLC), because the latter techniques imply extensive sample manipulation before analysis and are affected by the limited stability of the preformed derivatives.2 However, such RP-HPLC-based methods have the advantage of being accessible to most analytical laboratories, since they do not require expensive dedicated instruments. In addition, manufacturing of dedicated instruments is being halted, making the availability of validated pre-column methods even more important.In this paper, we describe the validation of a method that takes advantage of robotic sample derivatization, thereby limiting considerably the manual manipulation of samples. Another advantage of automation is that derivatization is performed just before the injection; therefore, the time from reaction to injection is kept absolutely constant for all samples, thus avoiding differential degradation of labile derivatives. We have studied the performance characteristics in terms of specificity, linearity, accuracy, precision, limit of detection, and limit of quantification for bovine serum albumin (BSA) and for a recombinant human Fab (rFab) fragment, whose extinction coefficient needs to be determined.Protein samples were hydrolyzed, then automatically derivatized with o-phthalaldehyde (OPA) and in-line analyzed by RP-HPLC with ultraviolet-visible (UV-Vis) detection, according to a method published in an Agilent application note.3.MATERIALS AND METHODSReagents, Solvents, and MaterialsSodium phosphate monobasic monohydrate, sodium hydroxide, boric acid, acetonitrile (LC grade), and methanol (LC grade) were obtained from Merck KGaA (Darmstadt, Germany). OPA reagent was prepared as described (Agilent art. 5061-3335, Palo Alto, CA). Borate buffer was prepared by adjusting 0.4 N boric acid to pH 10.2 with NaOH. Constant-boiling HCl was obtained from Sigma-Aldrich (St. Louis, MO). Chromatographic-grade water was produced by a Milli-Q system (Millipore, Billerica, MA)Disposable glass test tubes (50 × 6 mm) and hydrolysis reaction vials (25 × 120 mm) with Mininert valves were from Kimble Glass, Inc., and Kontes Glass Co. (Vineland, NJ). Amber wide-opening vials, glass conical inserts with polymer feet, and screw caps were from Agilent.Albumin standard solution (2 mg/mL) was supplied by Pierce Biotechnology (Rockford, IL), while amino acid standard mixtures at the concentration of 1 nmol/μL and 250 pmol/μL were from Agilent. The internal standard l-norvaline was obtained from Sigma-Aldrich. A recombinant Fab fragment (rFab) was obtained from the research laboratories of Bracco Imaging (Milan, Italy).Amino Acid Standard SolutionsAmino acid standard samples were prepared by mixing 95 μL of the 250 pmol/μL amino acid standard mixture with 5 μL of 10 mM norvaline and analyzed directly by RP-HPLC, within 24 h from preparation. Solutions for linearity study were prepared in duplicate by diluting the 1 nmol/μL amino acid standard solution, and contained 20, 50, 130, 250, or 500 pmol/μL of amino acid standard mixture together with 0.5 mM norvaline.Protein SamplesGlass test tubes (50 × 6 mm) were marked with incisions and soaked in a detergent solution for at least 12 h. They were rinsed thoroughly in Milli-Q water and dried in an oven at 80°C. Protein samples (7–75 μg) were transferred into the glass test tubes and spiked with 0.5 mM norvaline. They were quickly spun in a low-velocity centrifuge, then frozen and dried in a lyophilizer. Samples were then transferred into the reaction vial containing 0.5 mL of constant-boiling HCl on the bottom. Up to 12 test tubes could be accommodated in a reaction vial. The reaction vial was tightly closed and transferred into a pre-heated oven at 110°C for 18 h. The reaction vial was cooled at room temperature, then carefully opened under an aspirated hood. The test tubes were centrifuged and dried again in the lyophilizer to remove any liquid traces (condensed vapors). The dried residues were dissolved in 100 μL of 0.1 N HCl and transferred into the HPLC glass insert vials.InstrumentationAnalyses were performed using an Agilent 1100 Liquid Chromatograph, equipped with a binary pump delivery system (G1312A), robotic autosampler (G1313A), column thermostat (G1316A) and multi-wavelength detector (G1365A).Analytical ProcedureChromatography conditions were in accordance with the Agilent method.2 Briefly, the hydrolyzed samples and the norvaline-spiked amino acid standard solutions were automatically derivatized with OPA by programming the robotic autosampler (Table 1). After derivatization, an amount equivalent to 2.5 μL of each sample was injected on a Zorbax Eclipse-AAA column, 5 μm, 150 × 4.6 mm (Agilent), at 40°C, with detection at λ = 338 nm. Mobile phase A was 40 mM NaH2PO4, adjusted to pH 7.8 with NaOH, while mobile phase B was acetonitrile/methanol/ water (45/45/10 v/v/v). The separation was obtained at a flow rate of 2 mL/min with a gradient program that allowed for 1.9 min at 0% B followed by a 16.3-min step that raised eluent B to 53%. Then washing at 100% B and equilibration at 0% B was performed in a total analysis time of 26 min.TABLE 1Autosampler Programming InstructionsRESULTS AND DISCUSSIONAcid hydrolysis is a crucial step that considerably influences amino-acid recovery. In fact, during acid hydrolysis, tryptophan and cysteine are destroyed and serine and threonine are also partially lost, while methionine can undergo oxidation. Moreover, some amino acids such as glycine and serine are common contaminants; therefore, their quantification needs careful subtraction of average responses in blank runs, which, in the case of glycine, is also complicated by the fact that this residue is known to give rise to multiple derivatives after OPA reaction.2 Therefore, the validation parameters were estimated using the following seven best-recovered amino acids: Asx (Asn+Asp), Glx (Glu+Gln), Arg, Ala, Phe, Leu, and Lys.4In order to fully assess the method’s performance, both a standard amino acid mixture and a reference protein (e.g., BSA) should be assayed along with the product. The standard amino acid mixture (Figure 1​1)) enables the verification of the HPLC method’s performance, including derivatization, while the reference protein samples(Figure 2​2)) assess the completeness of the hydrolysis step. In addition, L-norvaline, which is added as the internal standard, provides a control for sample-to-sample variability.FIGURE 1Example of a standard amino acid mixture analysis at a concentration of 250 pmol/μl.FIGURE 2Example of a protein hydrolysate analysis for one of the 35-μg BsA samples.SpecificitySpecificity was documented by comparing retention times obtained in the standard amino acid mixture (five samples) with those obtained from the reference protein samples (three samples). Results are reported in Table 2. The minimal difference between retention times (