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 11))
enables the verification of the HPLC method’s performance, including
derivatization, while the reference protein samples(Figure 22))
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 (