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SUCCESSFULLY DEVELOPING AND USING SIZE-EXCLUSION CHROMATOGRAPHY FOR THE ANALYSIS OF BIOTHERAPEUTIC PROTEINS

Authors: Stephan M. Koza1, Pamela C. Iraneta, Priya Jayaraman

2, Erin E. Chambers

1.

1. Waters Corp., Scientific Operations, Milford, USA, 2. Waters Corp., Consumables Group, Milford, USA

INTRODUCTION Size exclusion chromatography (SEC) is a LC-based technique

commonly used to monitor protein aggregates throughout the

commercialization process of a biotherapeutic protein including

monoclonal antibodies. Traditional HPLC-based SEC methods

have used columns packed with >3 µm particles of an appropriate

pore size for the separation of proteins of different hydrodynamic

radii. Ideally, the proteins are separated solely on their relative

size in solution with larger aggregate species eluting prior to the

desire monomer drug.

This technical poster details the important instrument, method, and

SEC column use considerations in order to obtain robust, high

resolution, UHPLC-based SEC separations. It will also highlight

ways to substantially increase SEC column life that can be

adversely affected by injection of microbial and sample based

particulates.

LC SYSTEM PERFORMANCE The system performance attribute that has the greatest impact on the resolution of an SEC separation is extra-column dispersion. SEC is an isocratic separation with a k’ of effectively zero. Therefore, the impact that pre-column dispersion has on the separation quality is as impactful as that of post-column dispersion. Total extra-column dispersion can be measured using an experimental set-up as shown in Figure 1.

Method conditions:

– Mobile Phase: 3:7 Water:Acetonitrile at 0.3mL/min

–Sampling rate: 40Hz, λ = 273 nm

–Sample: 0.16 mg/mL Caffeine 1:9 Water:Acetonitrile ,1 µL injection

Extra-column dispersion was added pre-column through the use of sample loops ranging from 5µL to 30µL (10µL+ 20 µL). The extra-column dispersions for the system with the various sample loops added are show in Figure 2.

The dispersion volumes for the sample loops used were determined based on the cumulative relationship of the squares of the individual sources of dispersion:

The impact that increasing extra-column dispersion has on the UHPLC separations of monoclonal antibodies was evaluated for column lengths of 15 and 30 (2X15) cm.

SEC Method Conditions:

Flow rate: 0.35 mL/minute,

Mobile Phase: 20 mM Pi, 350 mM NaCl, pH 6.8

Samples: IgG1 mAb (infliximab) at 2mg/mL , Waters Intact mAb Mass Check Standard at 1 mg/mL, and IgG2 (panitumumab) at 20 mg/mL

Injection volumes: 1 and 2 uL for 150 and 300 mm columns, 0.1 µL for panitumumab experiments

Columns: Waters ACQUITY Protein BEH SEC, 200Å,

1.7µm, 4.6 X 150 mm and 300mm

Waters XBridge Protein BEH SEC, 200Å,

3.5µm, 7.8 X 300 mm

LC: Waters ACQUITY H-Class Bio

CDS: Waters Empower 3

MOBILE PHASE OPTIMIZATION In SEC, it is desirable for the separation to be wholly dependent on the hydrodynamic radii of the proteins being separated. As a results it is critical to develop a method in which hydrophobic and ionic interactions are kept to a minimum and the proteins and their covalent and/or non-covalent high molecular weight forms are fully recovered. An example of this type of method development is shown in Figure 6. By varying both pH and ionic strength poor recovery of the HMW and poor peak shape is observed at lower ionic strengths and higher pH. Operating this method at a pH of 7 and with 250 mM NaCl in the mobile phase would provide an adequate result however any minor deviation in pH or NaCl concentration could have a deleterious impact on the separation.

The subtle impact that a non-optimal mobile phase can have on HMW determinations was observed while evaluating an SEC method for the analysis of panitumumab, an IgG2 mAb

2. The effect that a non-optimal

mobile phase has on the recovery of the multimeric forms of panitumumab was observed upon repeated injections using a 20 mM sodium phosphate buffer at pH 6.8. In the replicate injections the level of the multimeric HMW peaks were observed to increase, however, after an overnight column flush at a 0.05 mL/min. flow rate the next injection saw multimer decrease to lower levels which then increased once again upon repeating (Figure 7). One explanation for this observation is that the multimeric forms are binding to the active sites on the column under these conditions and then either eluting slowly or dissociating and eluting. As shown in Figure 8, for a properly developed mobile phase (100 mm phosphate, 100 mM NaCl) the recovery of the HMW forms are consistent.

CONCLUSION LC system dispersion volume dictates the column ID size

requirements for SEC analysis.

Variations in dispersion can affect the quantitative result in

SEC. LMW results are more susceptible to error.

Non-optimal mobile phases can lead to poor quantitation of

components due to reduced recoveries of specific analytes.

References

1. Stephan Koza, Susan Serpa, Edouard Bouvier, and Kenneth J. Fountain, Successful Transfer of Size-Exclusion Separations between HPLC and UPLC, Waters .com, Library Number: APNT134822077, Part Number: 720005214EN

2. Fekete S, Ganzler K, Guillarme D. Critical evaluation of fast size exclusion chromatographic separations of protein aggregates, applying sub-2μm particles. Journal of Pharmaceutical and Biomedical Analysis. 2013; 78:141-9.

LC SYSTEM PERFORMANCE

(CONTINUED) Shown in Figure 3 is a comparison of the results for the high molecular weight (HMW) component of the Waters Intact mAb Mass Check Standard obtained with varying extra-column dispersion. While the quality of the separation decreases as indicated be the reduction in peak-to-valley ratio (P/V) the quantitative result for the percent area of the HMW forms is not significantly altered. A similar result is also observed for the 300 mm column length.

Alternatively, the effect that extra-column dispersion has on the SEC separation of low molecular weight (LMW) mAb fragments from the monomer is demonstrated in Figure 4. Here we observe that the resolution and quantitative result for the LMW1 fragment (mAb minus one FAb arm) is greatly impacted by extra column dispersion for the separation on the 150 mm column length while for the 300mm column length the effect is significantly reduced. This is in part the result of the greater initial resolution obtained with the longer column. Additionally, the resolution on the 300 mm column is also less impacted by extra column dispersion due to the greater peak volumes generated. In either

case, controlling extra-column dispersion to a minimum is a critical for the successful use of these UHP-SEC columns.

In situations where the extra-column dispersion of the available LC instrumentation cannot support the use of a 4.6X300 mm column it may be necessary to scale the method to a larger column diameter and particle size. When transferred correctly, matching L/dp and reduced linear velocity comparable results are obtained (Figure 5) albeit at 5X the analysis time

1.

Figure 1. Experiment for extra-column dispersion measurements using a zero-dead volume connector in place of a column

Figure 2. Results for extra-column dispersion measurements using a zero-dead volume connector in place of a column

Figure 3. The impact of increased extra-column dispersion on the reso-lution and integrated levels of mAb Mass Check Standard HMW using a 4.6 X 150 mm column packed with 1.7 µm particles.

Figure 4. The impact of increased extra-column dispersion on the reso-lution and integrated levels of infliximab LMW using a 4.6 mm ID col-

Figure 5. A comparison of separations of infliximab on two 3.5µm col-umns (300 mm length X 7.8 mm ID) run in series using an HPLC (Top Frame) and on a 1.7µm column (300 mm length X 4.6 mm ID) using a UHPLC (Bottom Frame).

Figure 6. An example of mobile phase optimization for SEC (pH and NaCl concentration). Mobile phases were blended from concentrated NaCl and phosphate buffers using AutoBlend + function in Empower 3.

Figure 7. An example of non-optimal mobile phase for SEC analysis of panitumumab. Inconsistent recovery of multimeric HMW (4.1 to 4.8min.) is observed.

Figure 8. Overlay of injection prior to 2 hr. column wash at 0.4 mL/minute and 3 subsequent injections using an optimal mobile phase for SEC analysis of panitumumab. Consistent recovery of multimeric HMW (4.1 to 4.8 min.) is observed.