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Rapid Measurement of Aggregation of Protein Therapeutics During Biomanufacturing


Protein therapeutics is the largest class of drugs made by the biotechnology industry. Aggregation of protein therapeutics is a major safety concern because aggregates can cause adverse, even life-threatening, immune responses in patients. The industry needs improved methods to detect, monitor, and understand the mechanisms governing aggregation during manufacturing. We are developing electrospray-differential mobility analysis (ES-DMA) to measure the size and 3D shape of aggregates of monoclonal antibodies (mAbs), the largest class of therapeutic protein. ES-DMA offer excellent potential for at line monitoring of protein size given its relative sample preparation and measurement times which are much shorter than typical unit process cycle times. Additionally, ES-DMA provides direct read-out of aggregate distributions with 0.1 nm resolution and does not expose samples to vacuum conditions.


Intended Impact

The intended impact of this work is to develop measurement methods and protocols to understand, monitor, and detect aggregation of protein drugs during biomanufacturing. These tools will enable a more fundamental understanding of the causes of aggregation, better strategies to limit or prevent it, and, ultimately, safer and more effective protein drugs.


  • Develop the measurement science and protocols for the application of ES-DMA as a near real-time bioprocess monitoring tool for protein aggregation.
  • Establish performance metrics for ES-DMA for measuring the aggregation of antibodies. Antibodies were chosen as a model system for these studies because they are the largest class of therapeutic proteins.

Technical approach

ES-DMA spectra where number density is plotted versus the mobility size in nm for (top) rabbit IgG at 18 mg/mL (120 nmol/L or 7.3 x 1013 antibodies/mL) in 20 mmol/L ammonium acetate, pH 8 and (bottom) human IgG previously lyophilized at approximately 75 mg/mL (500 nmol/L or 3.0 x 1014 antibodies/mL in 2.0 mmol/L ammonium acetate, pH 8.
   Electrospray-differential Mobility Analysis Figure 1
   Electrospray-differential Mobility Analysis Figure 2

In using ES-DMA to measure the size of protein aggregates, aqueous solutions of a protein are first introduced into the gas phase by electrospray. he various electrosprayed species are entrained in a gas flow into a differential mobility analyzer where they are separated according to their electrophoretic mobility. The DMA consists of two concentric cylinders where a voltage is applied to the inner cylinder to attract charged particles. Because the DMA operates at atmospheric pressure, protein species are subject to aerodynamic drag. When the proper voltage is applied to balance the aerodynamic and electrical forces exerted on a particle, the particle enters a collection slit where it is detected using a condensation particle counter. By sweeping the applied voltage, the DMA separates proteins based on their charge-to-aerodynamic size ratio (electrical mobility) and complete size distributions of species from 3 to 250 nm can be obtained. The DMA, in contrast to mass spectrometry, measures particle size so that species with molecular weights well in excess of 50 KDa can be characterized. For example, the instrument used in these studies can measure particles up to 120 nm in size corresponding to a molecular weight of ~400 MDa.
Both rabbit and human polyclonal immunoglobulins, IgGs, were characterized with ES-DMA and representative spectra are displayed at right. The relatively simple spectrum for the rabbit IgG is dominated by a large peak at 8.6 nm with a peak of much lower intensity appearing at 10.7 nm. In contrast, the spectrum of the human IgG is more complex with the most intense peak observed at 9.4 nm along with at least 5 peaks appearing at higher mobility sizes. The peaks at 8.6 nm for rabbit IgG and 9.4 for the human IgG are assigned to individual, intact antibodies. These mobility sizes represent the diameter of an equivalent sphere, exhibiting the same aerodynamic drag and charge as the antibody (i.e., electrical mobility). The mobility size measured with ES-DMA was compared with sizes calculated from protein crystallographic models. The atomic coordinates of the crystallographic structure of human IgG found in the protein database are used to calculate the projected area along 3 coordinates of the IgG molecule and from this an effective area of IgG and IgG aggregates (dimer, trimer, tetramer, etc.) is determined. Good agreement between measured and calculated sizes allow us to assign the higher mobility size peaks of human IgG to dimer, trimer, tetramer, and pentamer aggregates of IgG. We were also able to estimate the equilibrium constant for formation of the human IgG dimer by varying the solution concentration of the protein and measuring the change in size distributions using ES-DMA. The value of Keq determined, ~6.3 x 105 L/mol, is consistent with previously reported values for weakly associating proteins in solution, thereby validating ES-DMA as a method for measuring protein aggregation. In future studies, we will attempt to use ES-DMA to measure protein aggregation from crude cell culture extracts such as those found in a typical bioreactor for production of monoclonal antibodies.

Recent publications

Determination of protein aggregation with differential mobility analysis: Application to IgG antibody; Biotechnology and Bioengineering 101 (2008), 1214-1222.

Major Accomplishments:

  • Demonstrated that ES-DMA can be used to measure the size distribution of antibody aggregates of IgG.
  • Definitively assigned protein aggregate peaks measured by ES-DMA to structural models of IgG from the protein crystallographic data base.
  • Established the protein concentration and ionic strength ranges for using ES-DMA to measure protein aggregation.

End Date:


Lead Organizational Unit:



Leonard F. Pease III (University of Utah)
John T. Elliot (Biochemical Science Division)
Michael J. Tarlov (Process Measurements Division)
Michael R. Zachariah (Process Measurements Division/University of Maryland)


Michael J. Tarlov

301-975-2058 Telephone

100 Bureau Drive, MS 8362
Gaithersburg, MD 20899-8362

Michael R. Zachariah

301-975-3153 Telephone

100 Bureau Drive, MS 8360
Gaithersburg, MD 20899-8360