HPLC Method development for

Presented byS. Shanti KumarPA-Ph.D-2012-

103

Contents

Introduction1

Regulatory Aspects 2

Implementation of QbD3

Proposals of EFPIA/PhRMA Paper4

QbD- Analytical methods5

Benefits of QbD6

Challenges72

Pharmaceutical Quality in the 21st Century – A Integrated Systems Approach - Janet Woodcock

1. Follow preferred method development scheme and do “hands-on” method development – based on selectivity – changing parameters. e.g., pH, column or mobile phase types.2. Use method development software – run a few predictive runs and get prediction for best method.3. Evaluate multiple columns/multiple mobile phases in a manual or automated fashion, determine best results (e.g. using value switching to change between columns, software to track peaks etc.)

Practice #1 is used by many and incorporates “hands on” learning.It requires nothing special to make it work, so we will work through a method development scheme for this practice.

What are some standard method development

practices ….?

Bio-Separations : An Overview

Analyticalmethodologies

Separation &Purification Methods

HPLC

Capillary Electrophoresis

Mass Spectrometry

MethodologyMontage

Liquid-LiquidDistribution

DisplacementChromatography

Expanded-BedAdsorption

MembraneChromatography

SimulatedMoving Bed

Chromatography

Amino acids are the basic units of polypeptides and proteins. The primary amino acid sequence and tertiary structure (i.e.,

three-dimensional shape) are the most important features to consider when planning an HPLC separation.

Knowledge of the primary amino acid sequence allows for consideration of which functional groups are present in what quantity. Both amino acid side-chain and main-chain functional groups can interact with HPLC phases.

Knowledge of the protein's tertiary structure provides consideration of which functional groups are on the exterior of the protein. With a protein in its native conformation, surface side chains will have the greatest interaction with the HPLC support.

During the course of an HPLC separation, unfolding or denaturation of a protein or peptide may occur. Irreversible unfolding will lead to a loss of the protein's biological activity.

Unfolding can occur as the protein moves through the HPLC system, and during this unfolding, interior amino acid side chains will be exposed.

Often, the interior amino acids of a protein are predominantly hydrophobic and, during unfolding, additional interactions with non-polar stationary phases will occur, leading to increased retention and decreased separation efficiency. Unfolding can occur in different degrees, and the native conformation and unfolded conformations can have different retention times.

Various components of the chromatographic system can contribute to protein unfolding, including stationary phase hydrophobicity, mobile phase polarity, mobile phase pH, ion-pairing agents, detergents, and oven temperature.

The possibility of intermolecular interactions must be considered when unexpected peaks or low recovery occurs during analysis.

The hydrophobic amino acids are shielded from the solvent, providing stability to the structure.

Proteins and peptides can be analyzed by several HPLC modes, with reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC) the most common and the easiest for method development.However, ion-exchange chromatography (IEC) and size-exclusion chromatography (SEC) are also used to successfully separate and purify mixtures containing proteins and/or peptides.A comparison of these modes demonstrates the general principles of each technique.

RPLC and HIC are popular means of separating or purifying proteins and peptides. The hydrophobic character of proteins and peptides enables elution. Mixed hydrophobicity and charge characteristics of protein mixtures lead to non-ideal chromatographic behavior. HPLC methods tend to work well with small proteins and peptides, but suffer with larger or more unstable protein samples.Unfolding. The stationary phase support, ligates, surface tension, mobile phase polarity, temperature, pH, and mobile phase additives can all contribute to unfolding and may need to be adjusted during method optimization.Conformation changes due to unfolding are minimized at low temperatures and methods designed to prevent unfolding may be more successful at 25-30~ rSome protein unfolding is reversible. Partial unfolding that may occur during RPLC or HIC analyses does not necessarily mean bioactivity is lost. Both chromatographic modes have been used successfully to isolate biologically active proteins.

For RPLC or HIC method, parameters to be optimized includecolumn (stationary phase) choice, type and concentration of ion-pairing agent,organic solvent choice, pH, salt choice and concentration (HIC), mobile phase gradient, temperature.

1Column & S.P Choice

2Mobile phase

characteristics

3Choice of Ion pair

reagent

Particle size.For RPLC, sample loading capacity is related to the effective surface area available for interaction, a property related to the particle size of the packing, among other factors.Particle sizes of RPLC columns range from 1.5 to 25 µm. Band broadening can bea problem with large samples. HIC columns have particle sizes of 15-65 µm, and a larger sample can be accommodated, assuming other column factors including bonding density, type of bonding, porosity and stationary phase volume are equal.

Pore size can be chosen based on knowledge of molecular diameter. The minimum pore diameter that is acceptable for protein-ligate interaction is four times the protein's diameter. For beginning method development for a protein separation, choosing a pore size of greater than 30 nm will ensure acceptable diffusion of the large analyte into the pore. For peptide separation, a pore size of 10 nm is generally sufficient.

In RPLC, this is a water-organic eluent. Typically, aqueous-based mobile phases are used since non-aqueous phases contribute to protein denaturation.Often the mobile phase is at a low pH to stabilize ionizable side chains. A gradient mobile phase program that increases the concentration of organic is utilized for samples requiring separation of several components. Gradient programs running to 100% organic are common for pharmaceutical products that have complex matrices in order to ensure elution of compounds that associate strongly with the stationary phase. The gradient used in HIC is an increasing salt concentration in a buffered aqueous mobile phase.

IEC has found a home as the common technique for the separation of charged species.

IEC achieves separation of proteins and peptides by analyte-ligate electrostatic interactions.

Multiple charged functional groups.

Electrostatic interactions between ions in the solute, mobile phase, and stationary phase are the primary contributors to retention.

Elution of these biomolecules is complicated by the presence of multiple charged regions on a molecule.

In singly charged molecules, elution is linear in response to pH change. Multiple interactions, nonlinear with pH. The tertiary structure of proteins. Surface or surface accessible

ionizable groups.

Method development parameters in IEC include stationary phase choice mobile phase pH ionic strength choice of M.P ions temperature.

The IEC column consists of the stationary phase resin with immobilized strong or weak cation or anion exchangers. The degree of ionization differentiates a strong exchanger from a weak exchanger.Strong anion exchangers, for example, quaternary amine functional groups, or strong cation exchangers, such as sulfonated functional groups, are completely ionized.Separations of proteins and peptides employing strong exchangers are more straight forward. The exchange capacity of weak anion exchangers, such as primary, secondary, or tertiary amines, or weak cation exchangers, carboxylic acids, is variable.

The degree of ionization of weak exchangers changes as mobile phase pH approaches the pK a of the exchanger's functional group. The retention mechanism for IEC of proteins and peptides becomes very complicated with the use of weak exchangers. As a basic guide for: choosing a column, acidic proteins are analyzed on an anion exchange column, and basic proteins are analyzed on a cation exchange column. Most native proteins are negatively charged; consequently, anion exchange columns are used.

Elution in IEC occurs through increasing the ionic strength of the mobile phase. Thus, a gradient of increasing salt concentration is the minimum requirement for an IEC mobile phase. A small change in ionic strength can have a large effect on retention since many groups on a protein may be affected at one time. The choice of salt affects selectivity, although predicting the exact effect of any particular salt is difficult.

Small amounts of organic modifier maybe added to minimize hydrophobic interactions with the stationaryphase.

. SEC tends to be used for separation of high molecular weight species from biological matrices .

. It does not play an extensive role in identification or quantitation of proteins or peptides mainly because of the low resolution.

. Molecular hydrodynamic volume. . Hydrophobic interactions.. Protein unfolding. Ex. protein aggregation can lead to decreased SEC retention time.. Method development parameters

for SEC include buffer choice, salt concentration, organic modifier and flow rate.

. SEC columns.

Mobile phase optimization is the most difficult portion of SEC method development.

SEC mobile phases are generally neutral buffers with a salt concentration near 0.1 M. However, method development will likely require adjustment of pH and salt concentration.

The wide variety of possible protein and peptide combinations makes it difficult to suggest particular changes.

Not only does the hydrodynamic volume of proteins change with pH, mobile phase additives, or organic modifiers, but for peptide separation, a significantly different mobile phase composition may be required to achieve elution of different classes (e.g., acids, neutrals, etc.).

UV detection

PDA detection

Derivative spectroscopy

Fluorescence and electrochemical detection

The evaporative light scattering detector (ELSD).

It can offer increased sensitivity and multiple wavelength analysis.

Allows for peak purity to be assessed.

It is a very popular detection method for pharmaceuticals.

Large peak around 210nm, and a smaller peak around 280nm.

210-220nm due to the absorbance by the peptide bond.

Aromatic amino acid side chains absorb at 280 nm.

The prominence of computer-assisted analysis in pharmaceutical labs has led to derivative spectroscopy as a detection technique.

First, second, third, even fourth derivatives of the UV spectra can be plotted.

The resulting new minima or maxima offer additional information regarding the peak.

It is likely to find usefulness for methods with highly volatile mobile phases and lower-volatility high melting point analytes.

An investigation of HPLC protein and peptide separations using micro-bore HPLC columns was carried out by Issaq et al. The group looked at the effect of changes in column parameters and mobile phasecomposition on separation efficiency and selectivity. The group found improved sensitivity using smaller diameter columns andthe smallest pore size that is appropriate for the size of protein or peptidebeing analyzed. A tryptic digest of myoglobin showed maximum sensitivitywith a 0.18 mm i.d. column and silica pore size of 90 A. A 300 ~ipore size would be appropriate for analysis of larger proteins andpolypeptides.

Columns and the Separation of Proteins and Peptides

Several bonded phases, C18 , C8, C3, and CN, were testedfor their effect on a polypeptide separation. The polypeptides separated include Rnase, insulin, cytochrome C, lysozyme, pavalbumin, CDR, myoglobin, carbonic anhydrase, S-100b, and S-100A. Increasing the stationary phase alkyl chain length from C3-C 8 to C18 increased retention and resolution for all of the polypeptides except CDR and myoglobin.Those two analytes nearly co-eluted on the C18 column, co-eluted on theC 8 column, then reversed elution order and eluted well resolved on boththe C 3 and CN columns. Thus, the increased hydrophobicity of a longer alkyl chain can lead to changes in the three-dimensional shape of a protein that may account for the change in selectivity.

For the investigation of mobile phase conditions in the same study, a reversed-phase gradient was used since the majority of protein and peptide separations are carried out using RPLC. The basic gradient system was made of aqueous +0.1% TFA mobile phase A and organic +0.1% TFA mobile phase B with a linear gradient increasing in organic content.There was little difference in separation when acetonitrile and methanolwere compared as the organic modifier. The only difference in the separation of a mixture of five peptides (Gly-Tyr, Val-Tyr-Val, methionine enkephalin, leucine enkephalin, and angiotensin II) was improved resolution of the leucine enkephalin and angiotensin II peaks in the chromatogramwith methanol as the modifier.

Mobile Phase Conditions and the Separation of Proteins and Peptides

As the final concentration of organic in the linear gradient was increased (5-35%, 5-50%, 5-75%), retention decreased and efficiency increased. Selectivity changes occurred between leucine enkephalin and angiotensin II. At 5-35% acetonitrile, the peaks were resolved; at 5-50%, the peaks co-eluted; and at 5-75%, the peaks were resolved but the elution order was reversed. Several ionpairing agents were compared including TFA, heptafluorobutyric acid(HFBA), and formic acid.In the separation of a cytochrome C digest, TFA gave the best sensitivity, peak shape, and resolution. The effect of ion-pair agent concentration was also studied using the five-peptide mixture. A change in concentration of TFA affected peak height, selectivity, and resolution.An increase in TFA concentration improved peak symmetry andpeak width. The changes in resolution and selectivity did not follow atrend

A detailed series of studies on ion-pairing agents and HPLC peptideseparations was carried out by Hodges and coworkers.

They looked at the concentration of TFA and determined that increasing theconcentration of TFA to 0.20-0.25% increased sensitivity and separationof a mixture of peptides with similar charges.

The group also found that increasing the temperature from 25 to 70~ gave additionalimprovement in separation.

The same group compared four ion-pairing reagents (phosphoric acid, TFA, pentafluoropropionic acid (PFPA), and HFBA) at various concentrations for the effect on separating two peptide mixtures, one mixture containing peptides with the samenet charge, the other mixture containing peptides with varying net charge.

Ion-Pairing Agents and the Separation of Peptides

The researchers found that for both peptide mixtures, retention time increased with increasing concentration and hydrophobicity of the reagent.

Hydrophobicity had a larger affect on change in retention than concentration change. Increasing hydrophobicity of ion-pairing reagent also improved peak shape.

For peptides of identical charge, resolution was not affected by choice of ion-pairing reagent and was affected only slightly by an increase in concentration.

For peptides of varying charge, resolution increased with increased reagent concentration, although improvement in resolution diminished between 20 and 40mM for all reagents.

At 20mM, the best overall resolution was achieved with phosphoric acid, although phosphoric acid also showed the least change in resolution with an increase in reagent concentration.

Selectivity changes occurred with a change in reagent. The researchers also found that addition of a salt, such as sodium perchlorate (NaC104), to the mobile phase increased retention time and could improve peak shape.

Two research groups recently published methods to separate and quantitate phospholipids using HPLC with ELSD. Both methods separated a variety of phospholipids including phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), sphingomylelin (SM), and phosphatidic acid (PA).

Rombaut et al. TM utilized a 3.2 x 150mm, 3 gm silica column and a linear mobile phase gradient. The gradient began with 87.5:12:0.5 choloroform : methanol : buffer (1 M formic acid with triethylamine to pH 3) adjusting to 28:60:12 in 16 min. The buffer pH was found to affect peak shape and resolution of PS, PI, and PC.The acidic pH also increased column life. Several ELSD conditions were critical for detection of phospholipids. The optimal flow rate of the nebulizing gas (N2) was 1.4 mL/min and optimal nebulizing temperature was 85~ Increasing flow rate led to decreased response of all phospholipids. Increasing temperature had a similar but less pronounced effect. Detection limits for the phospholipids ranged from 11 to 40 ng.

The Separation and Evaporative Light Scattering Detection ofPhospholipids

Zhang et al. is used a 4.6 • 250 mm, 5 gm silica column with a gradient flow using a three-part mobile phase. An initial, isocratic composition of 42:5:53 isopropanol: water: hexane at flow rate of 0.8 mL/min was held for 8 min, then programmed to a final mobile phase composition of 54:10:36 from 8 to 25min at 1.2mL/min. Finally, an isocratic mobilephase composition of 66:17:17 was set from 25 to 35 min at 0.8 mL/min. The percent water in the mobile phase was the critical factor leading to elution and resolution of the phospholipids. ELSD conditions were 2 mL/min carrier gas (N2), drift tube temperature of 63.5~ and pressure of 0.4 MPa. This group determined that the drift tube temperature was the most important ELSD parameter during the analysis. Temperatures lower than 63.5~ led to baseline drift, and temperatures above 63.5~ led to decreased signal-to-noise ratio.

As a new type of chromatographic stationary phase, monoliths have been subjected to intensive study in the last years. They differ from other supports mainly in their characteristic structure, which results in the improved chromatographic properties.

Packed (a) and monolithic (b) chromatographic column. While most of the chromatographic supports are particle shaped,

monoliths consist of a single piece of highly porous material. In contrast to porous particle, the pores inside the monolith are open, forming a highly interconnected network of channels. Monoliths can be prepared in various ways and can have an inorganic or an organic based skeleton.

Silica-based monoliths The first being silica-based monolithic columns, generally prepared

using sol‑gel technology. This technology can be applied to create a continuous sol‑gel network throughout the column former by gelation of a sol solution within the capillary. Alternatively, it can be used to glue LC silica-based particles, once the capillary has been packed conventionally, producing a continuously bonded bed.

Organic polymer-based monolithsThe second category is rigid organic polymer-based monolithic columns, and these include acrylamide-based, methacrylate-based and styrene‑based polymers. The polymer network is generally formed inside the capillary by a step-wise chain polymerization reaction.Polymerization reaction mixtures usually consist of a combination of monomers and cross-linker, initiator and a porogenic mixture of solvents. A variety of monomers can be employed to fabricate the final monolith, being both charged and hydrophilic, to generate electroosmotic flow for capillary electrochromatography, or uncharged and hydrophobic, to allow reversed-phase interactions used in HPLC. The cross-linker concentration can be adjusted to change the degree of cross-linking which influences the overall porosity. An initiator is needed to begin the step-wise chain reaction, and it is often 2,2’‑azo‑bis‑isobutyronitrile (AIBN). The polymerization can be initiated using UV light or thermal treatment.Precipitation of the polymer occurs after it becomes insoluble in the reaction medium. Solubility is influence both the cross-linking and choice of porogen (a poor solvent for the polymer), which is commonly a mixture of alcohols.The formation of the monolith can be achieved in-situ within either untreated or pre-treated capillaries. The pre-treatment of the capillary often involves surface preparation for the introduction of double-bond functionality, allowing covalent bonding of the monolith to the capillary wall, which is of particular importance for HPLC application where the monoliths needs to withstand high pressures.Preparation of organic polymer monolithsThe polymerization mixture is forced into the capillary and generally initiated thermally. The reaction then continues by free radical polymerization to form a macroporous rigid monolithic polymer. The unreacted components, such as porogenic solvents, are then washed away.

1980’s

Hjerten

Hjerten introducedterm continuous polymer bed

for his compressed polyacrylamide gel.

7

macroporous poly(glycidyl

methacrylate-co-

-ethylene dimethacrylate) sheets

prepared by a bulk polymerization

process "macroporous

polymer membranes"materials

having similar

macroporous

structure and

cylindrical

shape

Continuous

polymer

rods1993

1990’s

First appearance of the term "monolith" to describe a single piece of functionalizedcellulose sponge used for the protein separation

Expression "monolithic" related to rigid macroporous polymers prepared by bulk polymerization in a closed mold has also been introduced and rapidly became a standard

Later

Historic Review

The popularity of these terms For example, BIA Separations is using their CIM (Convective Interaction Media) disks for separation processes they call "high-performance monolithic chromatography" since 1998. Recently, Merck K.G. commercialized their silica rod columns under the trade name Chromolith | in which theexpression monolith is also hidden.

Development of monolithic materials

Early attempts Hydrogels Researchers published the preparation of a continuous

polymer matrix and its use for the chromatographic separation in size-exclusion mode.

The column packed with the hydrogel particles afforded slightly better HETP of 0.88 mm but the selectivity was also rather poor.

The failure of this early approach most likely results from both unsuitable porous structure of the gel and column clogging by the action of hydrostatic pressure used to drive the flow.

Only Hjerten revisited the homogeneous swollen gels, used them in capillary electrochromatographic mode with electroosmosis as the flow driving force and achieved excellent separations.

Foams In 1970s, two groups

experimented independently with polyurethane foams prepared in situ within the confines of large chromatographic columns.

The Monsanto group adopted idea of shaping polymer foams and inserting them into a column. They extended the concept and prepared open pore polyurethane foam in situ.

They demonstrated that a decent gas chromatographic separations of various hydrocarbon mixtures could be achieved.

GC separation of C6-C9 alkanes on open pore polyurethane column.

The German group explored the polyurethane foams at about the same time demonstrated two techniques:

(i) production of a foam in situ and (ii) packing columns with the powdered foam. They expanded the arsenal of polymers and, in

addition to polyurethane, also used other foamed polymers such as styrene copolymers, natural rubber, and polyethylene.

Recent developments Compressed beds Prof. Hjerten knew from his early experiments

that beds of soft beads packed in chromatographic columns could be deformed to make the distances between the beads smaller thus achieving an enhanced resolution.

The major difficulty was to design such beds that simultaneously fulfilled two contradictory requirements in order to obtain the desired high resolution: a relatively low backpressure upon a strong compression of the beads.

Continuing in these efforts, his workers proposed an alternative approach, the gel was cut to small pieces, modified if desired, and its dispersion poured in a tube.

HPLC of model proteins in cation-exchange mode using compressed continuous gel at different indicated flow rates

This compression of the bed to only about 17 % of its original length resulted in a column enabling an excellent chromatographic separation of proteins in ion-exchange mode as demonstrated.

Monolithic disksSince objects in the bead shape were not

well suited for the measurements of mass transfer through the pores.

This led to the successful development of chromatographic media in a novel shape- flat disks.

However, soon thereafter, a small company BIA (recently renamed to BIA Separations) also noticed the advantages of the monolithic disk technology and got rapidly involved.

Quality by design and operations

Related Regulatory GuidanceRelated Regulatory Guidance

Facility OperationQbD based

Pharm.Quality

SystemsQ10

Risk Managemt.

Q9

Pharm.Develop.

Q8

8

TEXT TEXT

TEXT

Pharmaceutical

DevelopmentQ8

Collecting the

knowledge needed

Linkages – Q8, Q9 and Q10

TEXT

Pharm.Develop.

Q8

Risk Managemt.

Q9

Pharm.Quality

SystemsQ10

Quality RiskManagement

Q9

Application to our need using the

knowledge

Pharmaceutical

Quality System

Q10

Maintenance of our product,

process throughout the

product lifecycle9

DOE APPROACH

1Define the experimental design region

2Develop the

knowledge base

3Establish the design

space

Conduct formal experimental design Analyze results- built equations

Define best conditions Define process robustness

• Select the study variables

• Define the study ranges

The QbD Based methodology for development of robust analytical methods, V.Richard Verseput19

Formula : C10H13N5O4 Mol. Mass : 267.242 g/molCAS number : 30516-87-1Description : White to off-white crystals or needles.Solubility : Soluble in water (25 mg/mL at 25 °C) and ethanol (67 mg/mL)Optical rotation : +99° (c = 0.5 in water) Dissociation constant : pKa , 9.68 Spectroscopy data : Infrared, ultraviolet, nuclear magnetic resonance (proton) and mass spectral data have been reported

Zidovudine

Zidovudine is available as a 300-mg tablet, a 100- or 250-mg capsule, a 50-mg/5 mL syrup and a 200-mg/20 mL injection solution.The following impurities are limited by the British and European pharmacopoeias: 1-[(2R,5S)-5-

hydroxymethyl-2,5-dihydro-2-furyl]-5-methylpyrimidine-2,4(1H,3H)-dione.

1-(3-chloro-2,3-dideoxy-β-D-ribofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione.

triphenylmethanol.

ATP

24

Method Development

I level- tailing, symmetry, frontingII level- tailing less than 1.5

Comprehensive Exp. DesignBased on syst. scouting

To separate &quantify

Define the method intent

Perform the experimental design

Evaluate exp. Results & select final method

Perform risk ass. with rugged. & robust.

Define analytical method performance control strategy

Structured methodologies such as Fishbone diagram is implemented

System suitability criteria can be defined to manage risk and ensure the method delivers the desirable method attributes.

QbD approach to analytical RP-HPLC method development and its validation, ijpps-journal, Vol 3, Issue 1, 2011

25

Text

Txt

Txt

Text

Text

HPLC for Biomol. Is critical

Highly complex Protein mixtures

Purification processes

Functional groups

Degradation products

Tertiary structure

Knowledge and Experience Beats Blind Experimentation

Diagram

ThemeGallery is a Design Digital Content & Contents mall developed by Guild Design Inc.

TitleAdd your text

ThemeGallery is a Design Digital Content & Contents mall developed by Guild Design Inc.

Diagram

Text

Text

Text

Add YourTitle Text

• Text 1• Text 2• Text 3• Text 4• Text 5

Add YourTitle Text

• Text 1• Text 2• Text 3• Text 4• Text 5

Text

Text

Diagram

Concept

Add Your Text

Text

Text

Text

Text

Text

Text

Diagram

Add Your Text

Add Your Text

Add Your Text

Add Your Title

Diagram

Add Your Text Add Your Text Add Your Text

Text Text Text

Diagram

Add Your Title

Text

Text

Text

Text

Progress Diagram

Phase 1Phase 1 Phase 2Phase 2 Phase 3Phase 3

Text1

Text2Text3

Text4

Text5

3-D Pie Chart

Marketing Diagram

Add Your TextAdd Your Text

Add Your Title here

Text1Text1Text1Text1