Analysis and Purification of Polypeptidesby Reversed-Phase HPLC

Reversed-phase HPLC is a valuable tool for theanalysis and purification of proteins and peptides.It is effective in separating peptide fragments fromenzymatic digests, in the separation and purificationof natural and synthetic peptides, and in purifyingproteins as large as 100 kD for characterization.

Why is reversed-phase so widely used?

The answer is resolution. RP-HPLC is able toresolve very similar polypeptides, some of whichdiffer by a single amino acid residue. The separationof insulin variants (below) is an example of the highresolution capabilities of reversed-phase HPLC. Someinsulin variants differ by as little as a single aminoacid. For instance, rabbit insulin has a threonine wherehuman insulin has a serine—a difference of onemethylene group in a single residue—and RP-HPLCis able to separate these two variants. In anotherinstance, the purification of insulin-like growth factor(IGF), RP-HPLC separated native IGF from itsoxidized methionine derivative—a difference of asingle oxygen atom in a molecule of nearly 5800 MW.

The power and popularity of reversed-phaseHPLC is in its high resolving power!

Polypeptides are large molecules and cannot “partition” intothe stationary phase as do small molecules. Polypeptidesadsorb onto the hydrophobic “reversed-phase” surface fromthe mobile phase and remain adsorbed until the organiccomponent of the mobile phase reaches a criticalconcentration. At that time the polypeptide desorbs and iscarried by the mobile phase to the column exit. There is littlefurther interaction with the reversed-phase surface.Polypeptides elute quickly once the critical organicconcentration is reached, which accounts for the sharp peaksand high resolution achieved with this technique.X. Geng and F.E. Regnier, J. Chrom 296, 15-30 (1984).

Insulin variantsThe separation of insulin variants, some of which differby a single amino acid, demonstrates the ability ofreversed-phase HPLC to separate very similarpolypeptides.

ConditionsColumn: Vydac 214TP54 (C4, 5 µm, 4.6 mm i.d. x 250 mm).Eluent: 27-30% ACN with 0.1% TFA over 25 min.From J. Rivier and R. McClintock, J. Chrom. 268, 112-119 (1983).

rat II

rat I

rabbit

human

porcine

chicken

bovine

ovine

25 min0

Gradients in the organic modifier concentrationare normally used to obtain sharp peaks and optimumselectivity. Initial organic concentrations range from1-2% for hydrophilic peptides to 20-40% for large orhydrophobic proteins. While most peptides will elutein 70% organic modifier or less, large or hydrophobicproteins may require as high as 85% organic to elute.

The gradient slope (percent change in organicmodifier per unit volume or time) is normally around1-2% per minute, however very shallow gradients—aslow as 0.05-0.2% per minute—are used to separatecomplex mixtures or very similar peptides.

Polypeptides are usually detected by UV absorp-tion at wavelengths from 210 to 220 nm, where thepeptide bond absorbs. Higher wavelengths such as280 nm are sometimes used to monitor proteins witharomatic residues such as tryptophan.

Analysis and Purification of Polypeptides by Reversed phase HPLC

The Handbook answersmany questions commonlyasked about the reversed-phase chromatography ofproteins and peptides.

Available FREE from Vydac:

The 2nd edition of The Handbookof Analysis and Purification ofPeptides and Proteins byReversed-Phase HPLC

References1. The Importance of Silica Type for Reversed-Phase Protein

Separations, J.D. Pearson, N.T. Lin, and F.E. Regnier. Anal.Biochem, 124, 217-230 (1982).

2. Retention Model for Proteins in Reversed-phase Liquid Chroma-tography , X. Geng and F.E. Regnier, J. Chrom. 296, 15-30 (1984).

3. Reversed-phase High-Performance Liquid Chromatography ofInsulins from Different Species, J. Rivier and R. McClintock,J. Chrom. 268, 112-119 (1983).

4. Reversed-Phase Chromatography of Interleukin-2 Muteins ,M. Kunitani, P. Hirtzer, D. Johnson, R. Halenbeck, A. Boosman, andK. Koths, J. Chrom. 359, 391-402 (1986).

Separation conditions

Polypeptides are eluted from reversed-phasecolumns using aqueous mobile phases containing anion pairing agent and an organic modifier. The mostcommonly used ion pairing agents include:

Trifluoroacetic acid is by far the most commonlyused ion pairing agent because of its excellentseparation capabilities, low UV absorbance, and highvolatility for easy removal in peptide isolation. TFA isalso very effective in solubilizing hydrophobicpolypeptides.

HFBA is effective in the chromatography of basicproteins, and TEAA has shown unique selectivity for avariety of peptides.

The most commonly used organic modifiers are:❏ acetonitrile (ACN)❏ isopropanol (IPA)❏ ethanol (EtOH)

Acetonitrile offers low viscosity, excellent UVabsorption characteristics, and high volatility for easyremoval. Isopropanol is used either alone or incombination with acetonitrile (1:2 to 2:1) to elutelarge or hydrophobic proteins. Ethanol is most oftenused in large-scale process applications.

ACIDSAceticFormicPerchloricPhosphoricTrifluoroacetic (TFA)Heptafluorobutyric (HFBA)

BASESTriethylamine (TEA)Tetramethylammonium (TMA)Tetrabutylammonium (TBA)Triethylammonium acetate (TEAA)

Column Selectivity: The Primary Key toAchieving High-Performance HPLC

Separations of Proteins and Peptides

Keys to Well-Characterized Biotechnology ProductsRecent changes in FDA rules simplify the regulatory

treatment of “well-characterized biotechnologyproducts”*. HPLC often plays a crucial role in charac-terizing biotechnology products as to purity, potency,and identity.

Keys to defining a “well-characterizedbiotechnology product” by HPLC are:

✓ Selectivity: To monitor biotechnology products forpurity and identity: important impurities must beseparated from the major product, and digest fragmentswith minor changes must be resolved from normalfragments. Resolving minor impurities such as deami-dation products and oxidized methionine variants placethe ultimate demands on HPLC column selectivity.

✓ Stability: Column selectivity must be constantover hundreds of injections to ensure robust andreliable assays. Only columns which are physicallyand chemically stable and which maintain selectivityover time are practical for monitoring purity, potency,or identity of "well-characterized biotechnologyproducts".

✓ Reproducibility: Column selectivity and sampleresolution should remain the same when used columnsare replaced with columns from a new batch. HPLCcolumns used to monitor purity, potency, or identity of"well-characterized biotechnology products” must bereproducible from batch to batch.

Selectivity

Selectivity is the primary measure of columnperformance; a column must separate keyimpurities to be useful for an assay orpurification .

Selectivity and resolution of peptides and proteinsare very sensitive to column characteristics such as thesilica matrix and bonding chemistry. Vydac reversed-phase HPLC columns for bioseparations are madewith the 300 Å pore diameter silica that pioneered theseparation of polypeptides nearly two decades agoand continues to be the standard against whichall polypeptide separations are compared. Vydac'sunique polymeric bonding chemistry offers unrivaledselectivity for separating polypeptides.

Vydac reversed-phase columns have been used toseparate all types of polypeptides including nearlyidentical insulins (see Ref. 3 on p. 11), interleukinmuteins (see Ref. 4 on p. 11) and deamidated peptides(see figure below).

Vydac selectivity is illustrated in the figurebelow by the separation of two very similardigest peptide fragments where asparagine isconverted to isoaspartate by deamidation.

For details on how HPLC column selectivity can be optimized to best characterize your biotechnologyproduct, request Vydac Technical Report:

* The term “specified product” is favored for future use to eliminate thesubjective aspect of “well characterized.”

30 min

Separation of tryptic digestfragments from normal anddeamidated bovine somatotropin

modified(isoaspartate)

normal(asparagine)

Conditions:Column: Vydac 218TP54 (C18, 5 µm, 4.6 mm i.d. x 250 mm).Eluent: 0-15% ACN over 20 min., 15-2% ACN over 12 min.,21-48% ACN over 27 min., 48-75% ACN over 4 min.; in 0.1%aqueous TFA at 2.0 mL/min.From: Schlittler, et. al., Ninth ISPPP, Abstract 621 (1989). 0

Stability

Column stability is the second measure ofcolumn performance. Stable columns resultin constant column selectivity and sampleresolution over hundreds of sample injections,thus ensuring robust and reliable assays formonitoring purity, potency, or identity ofbiotechnology products.

HPLC column stability in polypeptide separationsrequires that columns retain their selectivity evenunder the somewhat harsh conditions of peptideanalysis – pH 2 or less with trifluoroacetic acid. Vydacreversed-phase HPLC columns have long been knownfor unusually long column lifetimes under thesedemanding conditions, often exceeding a thousandsample injections per column. The extended lifetimeof Vydac columns is attributed to the exceptionallystable silica matrix and to unique, polymeric bondedphases.

To show how column stability results inreliable assays for proteins and peptides, Vydactested a 214TP54 (C4, 5 µm, 4.6 mm i.d. x250 mm) column under extreme elution condi-tions of pH 1.5 with 0.5% trifluoroacetic acidat a temperature of 60°C – well outside theranges of normal polypeptide chromatography.Over 1000 gradient runs produced no changein column selectivity; resolution between allof the peptides and proteins tested remainedthe same – a remarkable indication of long-term column stability (see figures at right).

Keys to Well Characterized Biotechnology Products

Column stability test procedure:To provide an extreme test of column stability, over 1000 repeatgradients were run on a Vydac 214TP54 (C4, 5 µm, 4.6 mm i.d.x 250 mm) column under extreme conditions of pH andtemperature. Gradients were run from 0-100% B over 80minutes at 1.0 mL/min at a temperature of 60° C. Thestarting eluent (A) contained 0.5% TFA (pH 1.5) in water.The final eluent (B) contained 0.45% TFA in acetonitrile.The unusually high concentration of TFA (0.5%) and unusuallyhigh temperature (60° C) ensure an extreme test of columnstability. A test mixture consisting of six peptides (oxytocin,bradykinin, angiotensin II, eledoisin-related peptide, neurotensin,and angiotensin I) and three proteins (ribonuclease, insulin, andlysozyme) were run every 50-100 gradients under the testconditions. Results: The chromatographic separation of thepeptides and proteins is nearly identical after 1037 gradient runsto that obtained during the initial separation. The column didnot degrade, and selectivity and resolution remainedunchanged over 1037 gradient runs under the extremeconditions of pH and temperature used.

Initial separation of test mixture ofpeptides and proteins at beginnningof test

Separation of test mixture of peptidesand proteins after 1037 gradient runswith 0.5% TFA (pH 1.5) at 60o C.

Keys to Well Characterized Biotechnology Products

Reproducibility

Batch-to-batch column reproducibility is thethird measure of column performance. Repro-ducible HPLC columns are essential in thedevelopment of robust assays of biotechnologyproducts.

Column selectivity and sample resolution shouldremain the same when used columns are replaced withcolumns from a new batch. Columns which retainselectivity and separation characteristics from batch tobatch reduce hassle and ensure reliable assays over thelifetime of the biotechnology product.

Batch-to-batch column reproducibility depends onabsolute control over the manufacture of the silicamatrix and the bonding chemistry. Vydac manufac-tures HPLC silica in its own facility and maintainsabsolute control over the production process throughin-process quality control and Statistical ProcessControl measurements. These ensure highly reproduc-ible, high quality silica particles.

Vydac uses very sensitive tests to monitor andcontrol carbon loading on reversed-phase materials;tests which surpass conventional methods of measur-ing carbon loading such as carbon microcombustion.One such test used to monitor the carbon load ofpolymerically bonded C18

is based on a test developedby the U.S. NIST (National Institute for Standards andTechnology), involving the isocratic separation ofthree polyaromatic hydrocarbons and the calculationof a resolution factor, α, between two of these (seefigure). Vydac is able to precisely control the carbonload of polymerically bonded C18 through this highlysensitive test.

Vydac Application Note #9703, “Developing a Robust Reversed-PhaseMethod for Analysis of Polypeptides”, discusses the importance of reversed-phase HPLC in research, production, and quality control of polypeptidetherapeutics as well as its pivotal role in developing robust assays for well-characterized biotechnology-derived therapeutics. The publication emphasizesthe need for column stability and reproducibility and describes methods foroptimizing analyses by selection of ion-pairing agents, buffers, and eluant pH.

Vydac Application Note #9803, “Optimizing Peptide Purifications”, empha-sizes the importance of trial separations varying adsorbent type, pH, andbuffer ions in optimizing HPLC methods for routine purification of specificpeptides.

Column: 218TP5415 (C18, 5 µm, 4.6 mm i.d. x 150 mm)Eluent: 85:15 acetonitrile:waterSample: three polyaromatic hydrocarbons (NIST SRM 869)

Column reproducibility test procedure: The separation of peptide fragments from an enzymatic digestis a very effective test of batch-to-batch reproducibility ofreverse phase columns. A tryptic digest of β-lactoglobulin Awas chromatographed on Vydac 218TP54 (C18, 5 µm, 4.6 mmi.d. x 250 mm) columns from the following five lots: lot 930816-38-4 produced in January 1994; lot 931115-16-1 produced inMarch 1994; lot 940124-14-1 produced in September 1994; lot940415-20-1 produced in March 1995; and lot 950404-12-1produced in August 1995. Conditions were: 0-30% acetonitrilewith 0.1% TFA over 60 minutes at 1.0 mL/min. and 30°C.Conclusion: Vydac 218TP54 columns show remarkable lot-to-lot reproducibility.

Highly sensitive tests enable precise controlover reversed-phase carbon load and result inunparalleled batch-to-batch column reproduc-ibility for Vydac columns used in the analysisand purification of proteins and peptides.Batch-to-batch column reproducibility isevidenced by the separation of peptide fragmentsfrom the tryptic digest of β-lactoglobulin A.This digest was chromatographed on Vydac218TP54 columns (C18, 5 µm, 4.6 mm i.d. x 250mm) from several lots of materials producedover a period of more than a year (see figure).Digest maps performed on these columns werenearly identical—evidence of Vydac's excep-tional batch-to-batch column reproducibility.

Keys to Well Characterized Biotechnology Products

Lot 940415-20-1March, 1995

Lot 940124-14-1September, 1994

Lot 931115-16-1March, 1994

Lot 930816-38-4January, 1994

Lot 950405-12-1August, 1995