size exclusion chromatography

Illustrative description of separation in SEC

HONG, Paula; KOZA, Stephan; BOUVIER, Edouard SP. A REVIEW SIZE-

EXCLUSION CHROMATOGRAPHY FOR THE ANALYSIS OF PROTEIN

BIOTHERAPEUTICS AND THEIR AGGREGATES. Journal of liquid

chromatography & related technologies, v. 35, n. 20, p. 2923-2950, 2012.

FEKETE, Szabolcs et al. Theory and practice of Size Exclusion

Chromatography for the analysis of protein aggregates. Journal of

Pharmaceutical and Biomedical Analysis, 2014.

The free energy change of a chromatographic process can be described by, where ΔG 0, ΔH 0,and ΔS 0 are the standard free energy, enthalpy, and entropy differences, respectively; R is thegas constant: T is absolute temperature, and k is the partition coefficient. For mostchromatographic modes of separation, the enthalpy of adsorption is the dominant contributorto the overall change in free energy. SEC is unique in that partitioning is driven entirely byentropic processes as there ideally is no adsorption, ΔH = 0. Thus the previous equationbecomes: where K D is the thermodynamic retention factor in SEC. Thus, in SEC separations,temperature should have no impact on retention. In practice, temperature can indirectly impactretention to a small degree by altering the conformation of the proteins, as well as by affectingmobile phase viscosity and analyte diffusivity.

The thermodynamic SEC retention factor is the fraction of intraparticle porevolume that is accessible to the analyte: where V R , V 0, and V i are the respectiveretention volumes of the analyte of interest, the interstitial volume, and the intra-particle volume. K D will range from a value of 0 where the analyte is fully excludedfrom the pores of the stationary phase, to a value of 1 where the analyte fullyaccesses the intraparticle pores.

Separation of (1) thyroglobulin, (2) IgG, (3) BSA, (4) Myoglobin, and (5) Uracil on a Waters ACQUITY UPLC BEH200 SEC, 1.7 µ, 4.6 × 150 mm. Mobile phase: 100 mM sodium phosphate, pH 6.8. Flow rate: 0.3 mL/min. Temperature: 30°C (black), 40°C (blue), 50°C (red). Reproducedwith permission from Waters Corporation, Milford, MA

Typical SEC calibration curve

Theoretically expected impact of the particle size and mobile phase temperature on column performance. (For the calculations, a 50 kDa protein was assumed.).

Effect of linear velocity on plate height for (a) ribonuclease A (red) and (b) a monoclonalantibody (blue) on two columns varying in particle size. The 4.6 × 150 mm columns were packedwith either 1.7 micron (solid line) or 2.6 micron particles (dashed line). Pore size of stationaryphase sorbent: 200 Å. Mobile phase consisted of 100 mM sodium phosphate, pH 6.8.Reproduced with permission from Waters Corporation, Milford, MA.

Comparison of Columns: Effect of Particle Size on Efficiency and Resolution for a Reduced Antibody

Theoretical Plates

[-17pt] Columns

Dimensions (m i.d. ×

mm length)Particle

size (μm)Pore sizes

(Å) HC LC Resolution

TSKgel G3000SW

7.5 × 300 10 250 1980 3845 3

TSKgel G3000SWxl

7.8 × 300 5 250 5060 10674 4

Shodex KW-804

8.0 × 300 7 250 4952 8859 2

Protein-Pak 300SW

7.5 × 300 10 250 2078 4271 3

BioSuite 250

7.8 × 300 5 250 5149 9403 3

The drug aprotinin (Trasylol, previously Bayer and now Nordic Grouppharmaceuticals), is the small protein bovine pancreatic trypsin inhibitor,or BPTI, which inhibits trypsin and related proteolytic enzymes. Under thetrade name Trasylol, aprotinin was used as a medication administeredby injection to reduce bleeding during complex surgery, such as heart and liversurgery. Its main effect is the slowing down of fibrinolysis, the process thatleads to the breakdown of blood clots. The aim in its use was to decrease theneed for blood transfusions during surgery, as well as end-organ damage dueto hypotension (low blood pressure) as a result of marked blood loss. The drugwas temporarily withdrawn worldwide in 2007 after studies suggested that itsuse increased the risk of complications or death

Schematic representation of some of the key steps in non-native aggregation

Plate heights (HETP) vs. linear velocity (u0) plotsof Panitumumab (A), chicken ovalbumin (B) and β-lactoglobulin (C). Columns: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm (operated at 30, and60 °C), YMC-Pack-Diol-200 5 μm, 300 mm × 6 mm andPhenomenex Yarra SEC-3000 3 μm, 300 mm × 4.6 mm (operated at 30 and50 °C). Mobile phase: 20 mM disodiumhydrogen-phosphatebuffer of pH = 6.8.

Journal of Pharmaceutical and Biomedical Analysis 78–79 (2013) 141– 149

Effect of pressure (A) and temperature (B)on the observed aggregates. Column:Acquity UPLC BEH200 SEC 1.7 μm,150 mm × 4.6 mm. Mobile phase: 20 mMdisodium hydrogen-phosphate buffer ofpH = 6.8. Flow rate: 200 μl/min, detection:FL (Ex: 280 nm, Em: 360 nm). The columnpressure (head pressure) was varied byadding restrictor capillaries to the columnoutlet (131, 271, 406 and 465 bar weregenerated, including the column pressure)on (A). Sample: heat stressedpanitumumab.

Representative chromatograms on the effect of column temperature (A) and pressure (B) on the observed amount of antibody aggregates

Representative chromatograms on BEH 1.7 μm column (A) and on YMC diol 5 μm column (B) obtained by injecting the same sample (native panitumumab). Mobile phase: 20 mM disodiumhydrogen-phosphate buffer of pH = 6.8. Flow rate: 500 μl/min, detection: FL (Ex: 280 nm, Em: 360 nm), mobile phase temperature: 30 °C. The generated pressure was 274 bar (A) and 73 bar (B).

Fast separation of the aggregate and native form of β-lactoglobulin (A) and of chicken eggovalbumin (B). Column: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm. Mobile phase:20 mM disodium hydrogen-phosphate buffer of pH = 6.8. For β-lactoglobulin: flow rate:700 μl/min, mobile phase temperature: 45 °C and for egg ovalbumin: flow rate: 850 μl/min,mobile phase temperature: 60 °C. Detection: FL (Ex: 280 nm, Em: 360 nm) for both cases. Peaks:1, 2 and 3: high molecular weight species.

C. Wong et al. / J. Chromatogr. A 1270 (2012) 153– 161

SE-HPLC chromatogram profile at 280 nm showing fronting shoulder on monomer of a drug product at time zero (red) and 40 °C 2 months stability sample (black). Column TSKgel BioAsisstG3SWXL

Representative chromatograms of formulated bulk separation using 0.25 M NaCl (black), 0.5 MNaCl (blue), 0.75 M NaCl (green), 1.0 M NaCl (cyan), 1.25 M NaCl (magenta), 1.5 M NaCl(purple), and 1.7 M NaCl (red) sodium chloride in 20 mM sodium phosphate, pH 7.0 mobilephase, and Waters Acquity BHE200 4.6 mm × 300 mm column shown at 280 nm. (Forinterpretation of the references to color in this figure legend, the reader is referred to the webversion of the article.)

Representative chromatogram of (A) formulated bulk material (blue) and purified monomer fraction (black), and (B) 40 °C, 9 months stability sample (blue) and purified pre-peak fraction (black) using the final mixed mode UPLC method at 280 nm.

Mobile phase containing20 mM sodium phosphate atpH 7.0 and a flow rate of0.15 mL/min showed the bestseparation for the mixed modeUPLC method. The pre-peakand the monomer peak werefractionated using the finalmixed mode UPLC method forcharacterization

J. Sep. Sci. 2013, 36, 2718–2727

Chromatograms of PS standard (Mp ∼ 11 600 g/mol, -D- ∼ 1.03) obtained on XBridge(TM) C18 and ACQUITY R C18 columns (4.6 × 150 mm) packed with different size particles: 10, 5, 3.5, and1.7 m. Mobile phase, THF; flow rate, 1 mL/min; detection, UV at 254 nm.

Stacked chromatograms of SEC separation of two proprietary polymers on BEH 45 unbondedand BEH 45 TMS columns, 4.6 × 150mmin THF at 1 mL/min using UV detection at 254 nm. (A) Polymer A on BEH 45 unbonded; (B) polymer B on BEH 45 unbonded; (C) polymer A on BEH 45 TMS; (D) polymer B on BEH 45 TMS

Chromatograms of PSstandards on (A) BEH 200diol, 4.6 × 150 mm,detection, UV at 254 nm;(B) same as (A), but withELS detection. Fivereplicate injections areshown, demonstratingrepeatability; (C) 5 mPLgel MiniMix D (4.6 ×250 mm); detection, UVat 254 nm. In all cases,mobile phase was THFand flow rate was 1mL/min.

Chromatograms of (A) fourteencomponent mixture of PS standards obtained on two 4.6 × 150mm columns connected in series. First column, 1.7 m BEH diol 200 A° ; second column, 1.7 mBEH diol 450 A° . Mobile phase, THF; flow rate, 1 mL/min; detection, ELS, (B) twelve-componentPS standards obtained on three PL gel SEC columns (7.5 × 300 mm each) packed with 5 mPsdivinylbenzene particles with pore sizes labeled as 10E2, 10E3, and 10E4 A° . Mobile phase,THF; flow rate, 1 mL/min; detection, RI.

Chromatograms of PMMA standards and corresponding calibration curve (fifth-order fit). Mobile phase, THF; flow rate, 0.4 mL/min; column, 4.6 × 150 mm, packed with 1.7 mdiol-bonded particles with amean pore size of 200 A° ; detection, ELS.