Using Analytical Ultracentrifugation for Rapid Determination of Spectrophotometric Absorptivity

Spectrophotometric measurement of protein concentrations is a key analytical method for virtually all protein drug development and manufacture phases. For protein solutions, at least one initial, accurate protein concentration measurement is required to determine absorptivities (A0.1%) and molar extinction coefficients (ε).

Traditionally, dry weight analysis, quantitative amino acid analysis or total nitrogen analysis has been used to determine this crucial concentration, but all these methods are laborious, may require a relatively large sample size, and can yield unreliable results.

It has been suggested that the refractive increment of a protein may be applied universally for protein concentration measurement as it is relatively insensitive to its amino acid composition.

Previously, protein concentration has been determined using an analytical ultracentrifuge equipped with interference optics.

This article discusses the application of Beckman Coulter’s Optima XL-I analytical ultracentrifuge equipped with Raleigh interference and UV absorbance optical systems to rapidly determine the absorptivity for a recombinant IgG monoclonal antibody.

It compares the analytical ultracentrifuge data for determination of spectrophotometric absorptivities with the experimental data obtained from quantitative amino acid analysis and an enzymatic digestion method.

Materials and Methods

Monoclonal Antibody

Protein Recovery Sciences, Genentech supplied the monoclonal antibody. The sample was dialyzed to equilibrium in an appropriate formulation buffer and diluted with the solvent to an optical density range of 0.8-1.2 at a 280 nm wavelength.

Quantitative Amino Acid Analysis

The antibody concentration was determined using quantitative amino acid analysis in conjunction with the theoretical amino acid composition (based on expected composition from cDNA).

The absorptivity was then calculated using this concentration. The sample analysis was performed on two different days and triplicates were run on each sample.

Enzymatic Digestion Procedure

Protein concentration was also determined using a modification of Bewley. An AVIV 14DS UV-VIS spectrophotometer was used to measure the absorption spectra of the protein in formulation.

The process used four matched 1-cm cells. The reference cell contained, 1 mL of placebo, while the three sample cells contained 1 mL monoclonal antibody solution.

An absorbance between 0.8-1.2 absorbance units was produced by adjusting the protein concentration (unknown) with a buffer. Initially, the absorption spectrum of native antibody was obtained from the difference between reference and sample spectra.

Next, each of the antibody samples were digested in the sample cell through acidification with 0.06M HCl and addition of Aspergillus acid proteinase and pepsin. The reference cell was treated in exactly the same way.

Following complete digestion at room temperature (4-12h), spectra were measured. A log-log procedure was used to correct all absorption spectra for light scattering. AVIV Associates software was used to obtain second-order spectra from the zero-order data.

Rapid and Sequential Measurement of Absorption and Refractive Increment

An Optima XL-I analytical ultracentrifuge with integrated absorbance and Rayleigh interference optics was used to measure absorption spectra and an interference fringe pattern of the native antibody.

The reference (400μL) and antibody (190μL) were placed in a synthetic boundary cell with a 1.2-cm path. All absorption and fringe displacement measurements were carried out in the same cell in about 10 minutes at 20°C and a rotor speed of 3000 rpm. The detection wavelengths for absorption and fringe displacement were 280 nm and 670 nm, respectively.

Results and Discussion

Quantitative Amino Acid Analysis

The inter-assay and intra-assay variability that may arise when quantitative amino acid analysis is used for determining protein concentration is presented in Table 1.

The two monoclonal antibody absorptivity values obtained by this procedure were 1.71 ± 0.03 and 1.17 ± 0.22 mL/mg/cm. In addition to such variable results, a turnaround time of at least 24h is required for digestion and analysis.

Table 1. Absorptivity determined by quantitative amino acid analysis of monoclonal antibody

ASSAY #1

MEAN (N = 3)

Protein Conc. (mg/mL)

0.97

0.97

0.94

0.95 ± 0.02

A0.1% (mL/mg/cm)

1.68

1.73

1.72

1.71 ± 0.03

ASSAY #2

MEAN (N = 3)

Protein Conc. (mg/mL)

1.14

1.50

1.62

1.42 ± 0.25

A0.1% (mL/mg/cm)

1.43

1.08

1.01

1.17 ± 0.22

Enzymatic Digestion Procedure

The enzymatic digestion procedure is based on the classic hyperchromic effect of polypeptide folding on a protein’s UV absorption spectra. Figure 1 shows the absorption spectra of the antibody before and after proteolytic digestion.

When the protein conformation is destroyed, the spectrum undergoes the well-known denaturation “blue shift,” including a loss of absorbance. A model UV spectrum was obtained from the monoclonal antibody’s amino acid composition and represents the absorptivity of the protein in the absence of any conformational effects.

This model spectrum is a linear combination of experimentally determined spectra of the model compounds glutathione, N-acetyl-L-Phe-ethyl ester, N-acetyl-L-Tyr-amide, and N-acetyl-L-Trp-amide.

Zero-order absorption spectra of monoclonal antibody

Figure 1. Zero-order absorption spectra of monoclonal antibody before and after proteolytic digestion.

Additional data of absorbance inflections as a function of wavelength is provided by the second derivative of a UV spectrum.

The second derivative spectra of proteolytically digested antibody is almost identical to that of the model UV spectrum (Figure 2), strongly suggesting that the proteolytic digestion was adequate to eliminate any impact of protein structure on the protein UV absorption spectrum and the calculated model absorptivity of 1.509 at 275.7 nm should be equivalent to the digested sample.

It is possible to relate the absorptivity of a 0.1% solution of the native folded protein at the maximum absorbance wavelength (AN) to the model absorptivity (Amodel) and the absorbance before (Abspredigest) and after digestion (Absdigest):

AN = AbspredigestAmodel/Absdigest

Using this method, the absorptivity of native rhuMAbE25 at 278.4 nm was found to be 1.543 ± 0.004 (mg/mL)-1cm-1 for triplicate samples.Second-order absorption spectra of antibody

Figure 2. Second-order absorption spectra of antibody before and after proteolytic digestion.

Analytical Ultracentrifuge Procedure

A rapid method for measuring absorptivity uses the analytical ultracentrifuge featuring one spectrophotometric and one interferometric optical system. The absorption as a function of radial position at 280 nm is shown in Figure 3.

The mean value of the baseline was subtracted from the plateau to determine the absorbance of monoclonal antibody. This absorption value has not been further corrected for light scattering.

The fringe displacement as a function of radial position at 670nm can be seen in Figure 4. The mean value of the baseline was subtracted from the plateau to determine the fringe displacement (ΔJ) of 3.32 ± 0.035 fringes for the antibody. The following expression relates the protein concentration (C) to the measured fringe displacement:

C = DJ/k

where k = the specific fringe displacement for a cell optical pathlength (L) defined by: k = (dn/dc)L/λ.

For human γ-globulin, the specific refractive increment dn/dc was estimated to be 0.187 (g/mL)-1 at 670 nm, the wavelength (λ) of the laser light source used in the Optima XL-I refractive optical system at a temperature range of 20-25°C.

These values result in a computed value for k of 3.31(mg/mL)-1, which is used to convert the measured fringe displacement (ΔJ) value into a concentration value.

The concentration value in combination with the measured absorbance at 280nm of 1.89 ± 0.28 yielded an absorptivity of 1.57 ± 0.03(mg/mL)-1cm-1, which agrees well with the value determined by enzymatic digestion. Determined from sedimentation equilibrium, the molecular weight of 148kDa results in a molar extinction coefficient of 2.32 x 105 M-1cm-1.

Radial scanning of antibody at 20°C by the Optima XL-I UV absorbance scanner.

Figure 3. Radial scanning of antibody at 20°C by the Optima XL-I UV absorbance scanner.

Radial scanning of antibody

Figure 4. Radial scanning of antibody at 20°C using the Optima XL-I interference optical system.

Conclusion

This article has demonstrated the ability of the Optima XL-I analytical ultracentrifuge equipped with the integrated UV and interferometric optical systems to rapidly and accurately determine absorptivity and molar extinction coefficients.

It took less than 10 minutes to perform the entire procedure, which resulted in an absorptivity value that was in excellent agreement with the value determined by the enzymatic digestion procedure.

The values determined by the more traditional quantitative amino acid analysis varied and ranged from 1.01 and 1.73 (mg/mL)-1cm-1. The values lower than the predicted model value of 1.509 (mg/mL)-1cm-1 are probably inaccurate, as the model value is that anticipated for a fully unfolded protein.

The use of an immunoglobulin for this study was based on the fact that dn/dc values determined for γ-globulins by different labs are consistent once corrected for wavelength dependence using the Pearlmann and Longsworth method.

In this methodology, a critical assumption is that the specific refractive increment for proteins is virtually independent of amino acid composition. Inspection of refractive increment tables shows that this is correct in general, but that there can be significant variability, some of which may be due to errors in protein concentration determination and some of which may be due to contributions from carbohydrate.

A systematic analysis of the impact of carbohydrate and amino acid composition on the dn/dc may mean the analytical ultracentrifuge eventually becomes considered the method of choice for rapidly and accurately determining protein absorptivity and molar extinction values.

Alternatively, it should be possible to calculate refractive increment. This article has shown it is possible to calculate the refractive index from partial specific volume, amino acid composition and refraction per gram amino acid residue.

As previously suggested, this methodology needs to be extended to contributions from prosthetic groups such as heme and carbohydrates.

Acknowledgement

Produced from articles authored by R. A. Gray, A. Stern, T. Bewley, and S. J. Shire Genentech, Inc. South San Francisco, CA. Thanks to Mike Molony and Reed Harris, Medicinal Analytical Chemistry Department of Genentech, Inc., Paul Voelker and Don McRorie of Beckman Coulter Instruments, Inc., and Dr. Rodney Pearlman for their support on this project.

References

  1. Babul, J., Stellwagen, E. Measurement of protein concentration with interference optics. Anal. Biochem. 28, 216-221 (1969).
  2. Schachman, H. K. Is there a future for the ultracentrifuge? Analytical Ultracentrifugation in Biochemistry and Polymer Science, pp. 3-15. Edited by S. E. Harding, A. J. Rowe, and J. C. Horton.Cambridge, Royal Society of Chemistry, 1992.
  3. Bewley, T. A. A novel procedure for determining protein concentrations from absorption spectra of enzyme digests. Anal. Biochem. 123, 55-65 (1982)
  4. Winder, A. F., Gent, W. L. G. Correction of light-scattering errors in spectrophotometric protein determinations. Biopolymers 10, 1243-1251 (1971)
  5. Fasman, G. D., ed. CRC Handbook of Biochemistry and Molecular Biology, 3rd, Proteins, Vol. II, p. 376. Cleveland, Ohio, CRC Press, 1976.
  6. Van Holde, K. E. Physical Biochemistry, pp. 94-95. Englewood Cliffs, NJ, Prentice Hall, Inc., 1971.
  7. Pearlmann, G. E., Longsworth, L. G. The specific refractive increment of some purified proteins. J. Am. Chem. Soc. 70, 2719 (1948)
  8. McMeekin, T. L., Wilensky, M., Groves, M. L. Refractive indices of proteins in relation to amino acid composition and specific volume. Biochem. Biophys. Res. Commun. 7, 151-156 (1962)

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Last updated: Mar 1, 2019 at 5:13 AM

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