Threading the Molecular Needle: The Importance of Particle Pore Diameter for Biomacromolecule Separations

Stephanie Schuster, Application and Quality Manager, Advanced Materials Technology, Inc., Wilmington, DE;
Cory E. Muraco, Senior Scientist, Liquid Separations R&D, MilliporeSigma

Introduction

Recently, the evolution of middle to large biomacromolecules such as peptides or proteins is apparent in the market of pharmaceutical drugs in addition to traditional active ingredients based on small molecules. The use of peptides for clinical purposes has its origins in the 1920’s using insulin and penicillin. In the 1960’s, oxytocin and vasopressin were added to the clinician’s arsenal. Leuprorelin and octreotide rounded out the first 60 years of peptide-based drugs by being introduced to the clinic in the 1980’s.1

In the 1980’s, the average length of therapeutic peptides was nine amino acids long with molecular weights of less than 5 kDa. But in the 1990’s, longer peptides containing 15 – 20 amino acids became common, and, by the turn of the century, proteins with 40 to 50 amino acid residues with molecular weights approaching or exceeding 50 kDa were beginning to emerge en masse from biopharmaceutical companies. In 2019, even larger proteins have taken the market by storm in the form of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and bispecific antibodies (bsAbs), to name just a few.

A mAb is composed of two light chains (LC) that are tethered to two heavy chains (HC) through disulfide bonds. In addition, since the LC and HC are composed of amino acids with reactive side chains, IgG’s can be post-translationally modified through phosphorylation, methylation, oxidation, and nitrosylation, among other modifications. These modifications may change the binding affinity of the mAb so that it binds either the wrong antigen, does not bind any antigen, or associates with the wrong cell surface receptor. In addition, mAbs can also aggregate which can lead to allergic responses in patients. Biopharmaceutical companies need to develop rigorous methods to assess lot-to-lot reproducibility of their candidate biologic drug, and the above-mentioned modifications are known as Critical Quality Attributes (CQAs) that both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) monitor. Due to these stringent requirements from regulatory bodies, much research has been pursued in the past 20 years to develop accurate, robust, and high-throughput methods to assess biopharmaceutical purity and structure.

High performance liquid chromatography (HPLC) has been used extensively in the past several decades to characterize peptides and proteins. Since the advent of ultrahigh performance liquid chromatography mass spectrometry (UHPLC-MS) in the mid-2000’s, there has been much research in both developing new methods to characterize large biomolecules and in developing new column technology to better resolve all the different molecular entities that are present in a heterogeneous mAb therapeutic. Two main types of particle morphology are prevalent in the industry today: fully porous particles (FPPs) and superficially porous particles (SPPs, also called Fused-Core® or core shell particles). To take advantage of the low dispersion of UHPLC instrumentation, columns with sub-2 μm FPPs with pore sizes of 300 Å have been used for the analysis of larger hydrodynamic radii biomacromolecules. These columns have been the industry standard since the mid 2000’s. However, these columns suffer limitations when analyzing larger or more complex proteins like mAbs and antibody-drug conjugates (ADCs). The relatively small pore size, in addition to a totally porous architecture, and overall higher surface area, restricts the free diffusion of large molecules through the particle and may cause irreversible adsorption of the protein to the stationary phase. This architecture concomitantly results in an increase in the mass transfer term and longitudinal diffusion term of the Van Deemter equation, leading to peak tailing, loss of resolution, and low recovery.

Since 2017, a new line of 1000 Å columns have been introduced that have been optimized for mAb and ADC characterization. These columns are packed with 2.7 μm SPPs that are composed of a 0.5 μm shell thickness and a 1.7 μm solid silica core. The 1000 Å pore particle permits the analysis of mAbs, ADCs, and other, much larger, biomacromolecules. Advantages over columns packed with FPPs are numerous: the SPP shows a significant advantage in mass transfer, leading to less band spreading; columns packed with SPPs are more uniformly packed than columns composed of FPPs, leading to a lower eddy dispersion (A term in the Van Deemter equation); and larger particle sized SPPs have efficiencies similar to or better than sub-2 μm FPPs, leading to the ability of the analyst to run at higher flow rates with less risk of on-column frictional heating due to elevated column backpressure. Finally, the B-term (longitudinal diffusion) of the Van Deemter equation is also minimized with SPPs. This is due to the presence of less dead volume in the column. A column packed with FPPs will occupy only 33 % of the column volume whereas a column packed with SPPs will occupy approximately 41 % of the column volume.2

This article will further detail the reasons on why wide-pore diameters are necessary for separating biomacromolecules. Applications involving biomolecules will clearly demonstrate that pore diameters of 450 Å or less can lead to poor results and inadequate resolution of biomolecule variants.

Experimental and results

All the BIOshell™ IgG 1000 Å SPP columns were obtained from MilliporeSigma*. The experiments were conducted using Shimadzu Nexera X2 UHPLC instruments with PDA detection. Proteins and mAbs were obtained from MilliporeSigma. NISTmAb was purchased from NIST (Gaithersburg, MD). Trifluoroacetic acid was from Pierce Chemicals (Rockford, IL). Acetonitrile was from MilliporeSigma (Gibbstown, NJ). Difluoroacetic acid was purchased from SynQuest Laboratories (Alachua, FL).

Larger pores enable improved access to the stationary phase. Figure 1 shows the comparison of a separation of four proteins using a BIOshell IgG 1000 Å C4 column to a FPP 300 Å C4 column. Increased retention is observed on the BIOshell IgG C4 column indicating better access to the pores/stationary phase. The other advantage of the BIOshell IgG C4 column is that it provides narrower peak widths for all the proteins in the mix. This is especially noticeable for the largest protein in the mix – enolase. The peak width is 32 % narrower on the BIOshell IgG C4 column compared to the FPP 300 Å column.

 

Figure 1. Comparison of 1000 Å SPP and 300 Å FPP

Column:  As indicated; 15 cm x 2.1 mm I.D.
Mobile Phase:  [A] Water (0.1 % DFA); [B] Acetonitrile
(0.1 % DFA) 
 
Gradient:  23 % B to 50 % B in 24 min
Flow Rate:  0.5 mL/min  
Column Temp.:  60 °C
Detector:  UV, 280 nm
Injection:  1.5 μL
Sample:  Protein mixture, varied concentration, water

Comparison of 1000 Å SPP and 300 Å FPP

* Click on image to enlarge.

Another advantage of BIOshell IgG 1000 Å C4 columns is how well they can resolve isoforms of IgG2 mAbs. These mAbs differ in the arrangement of the disulfide bridges that connect the heavy and light chains in the hinge region. In Figure 2, six different isoforms of panitumumab (trade name Vectibix) are resolved on the BIOshell IgG 1000 Å C4 column whereas the FPP 300 Å C4 column is only able to resolve approximately two isomers. In addition the back pressure is only 120 bar compared to 205 bar on the FPP 300 Å C4 column. Similar resolution advantages are found when the BIOshell IgG 1000 Å C18 column is compared to a 3 µm FPP 300 Å C18 column as shown in Figure 3. In this example, resolution of minor components at the base of the main NIST mAb peak are revealed when the BIOshell IgG 1000 Å C18 column is used. Conversely, none of these minor peaks are visible in the separation when the separation is run on the 3 µm FPP 300 Å column C18 column.

 

Figure 2. Improved Resolution of IgG2 Variants

Column:  As indicated; 15 cm x 2.1 mm I.D.
Mobile Phase:  [A] 88:10:2 Water:Acetonitrile:n-Propanol (0.1 % DFA); [B] 10:20:70 Water:Acetonitrile:n Propanol (0.1 % DFA)
Gradient:  14 % B to 24 % B in 20 min
Flow Rate:  0.2 mL/min 
Column Temp.:  80 °C
Detector:  UV, 280 nm
Injection:  2 μL
Sample:  Vectibix, 2 mg/mL, water (0.1 % DFA)

Improved Resolution of IgG2 Variants


* Click on image to enlarge.

Figure 3. mAb Separation: 1000 Å SPP vs. 300 Å FPP

Mobile Phase:  [A] Water (0.1% TFA);
[B] Acetonitrile (0.1 % TFA)

Gradient:  30 % B to 45 % B in 15 min
Flow Rate:  0.4 mL/min 
Column Temp.:  40 °C
Detector:  UV, 280 nm
Injection:  2 μL
Sample:  Trastuzumab, 2 mg/mL, water (0.1 % TFA)

mAb Separation: 1000 Å SPP vs. 300 Å FPP

* Click on image to enlarge.

Increased temperature is often used as a means of increasing sample recovery when conducting protein analysis. What has been observed with BIOshell IgG 1000 Å Diphenyl phase is that recovery is equally sufficient at 40 °C as it is at 80 °C. In Figure 4, a separation of trastuzumab under 40 °C conditions is compared to a separation on a SPP 450 Å Polyphenyl column. Not only is the peak area greater with the novel BIOshell IgG 1000 Å Diphenyl column, but the resolution and retention are greater, too.

Sample mass on column has always been an experimental variable that chromatographers have had to consider. This variable is further magnified due to the slow diffusion kinetics of large molecules. Column overload can be monitored by observing an increase in peak width as an increasing amount of sample is injected onto the column. Figure 5 shows the advantages of using a wider pore stationary phase material in terms of band broadening. Much lower peak widths are observed with the BIOshell IgG 1000 Å column than a 300 Å FPP column.

 

Figure 4. mAb Separation: 1000 Å SPP vs. 450 Å SPP

Column:  As indicated; 15 cm x 2.1 mm I.D., 2.7 μm
Mobile Phase:  [A] Water (0.1% TFA); [B] Acetonitrile
(0.1 % TFA)
Gradient:  30 % B to 45 % B in 15 min
Flow Rate:  0.4 mL/min 
Column Temp.:  40 °C
Detector:  UV, 280 nm
Injection:  2 μL
Sample:  Trastuzumab, 2 mg/mL, water (0.1 % TFA)




* Click on image to enlarge.

Figure 5. Dependence of Sample Mass on Peak Width

Column:  As indicated; 15 cm x 2.1 mm I.D., C4 Phase
Mobile Phase:  [A] Water (0.1% DFA); [B] Acetonitrile
(0.1 % DFA)
Gradient:  27 % B to 37 % B in 10 min
Flow Rate:  0.5 mL/min 
Column Temp.:  80 °C
Detector:  UV, 280 nm
Injection:  0.1, 0.5, 1, 5, 10, and 20 μL
Sample:  mAb (trastuzumab), 7 mg/mL, water


* Click on image to enlarge.

Conclusion

Biomacromolecules are complex species requiring cutting-edge chromatographic materials and methods for full characterization. One of the principle parameters of the stationary phase that must be considered when attempting to analyze biomolecules is the pore diameter of the particle. If the pore diameter is too small, steric exclusion will occur resulting in the molecule not being able to access all the available surface area within the pore of the particle. This leads to low chromatographic performance of the method. Combining the high efficiencies garnered from the SPP core-shell architecture with a 1000 Å pore diameter, biomolecules can be completely characterized at a topdown level without fear of steric hindrance or exclusion.

* The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.

 

References

  1. Ikegami, T.; “Hydrophilic Interaction Chromatography for the Analysis of Biopharmaceutical Drugs and Therapeutic Peptides: A Review Based on the Separation Characteristics of the Hydrophilic Interaction Chromatography Phases.” J. Sep. Sci. 2019, 42, 130.
  2. Muraco, C. E.; “Improved Biomacromolecule Separations Using Superficially Porous Particles with a 1000 Å Pore Diameter.” Chromatography Today, 2018, 4, 27.

 

Materials