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Reversed Phase HPLC for the Analysis of Biomolecules

Biopharmaceuticals offer great hope in treating medical conditions which are currently poorly served, at best, by traditional pharmaceuticals. 

It is estimated that there are over 400 biopharmaceuticals in clinical trials for in excess of 200 disease areas.  The enhanced complexity and variability that comes from the size of biopharmaceuticals, allied with the intricacy of the production process, mean chromatography is employed to a much greater extent during production and release testing.  

Commonly reversed phase (RP), hydrophilic interaction (HILIC), ion exchange (IEX), and size exclusion (SEC) chromatographic modes will be combined to allow full characterization of these complex biomolecules. 

The following article will introduce the fundamentals of biopharmaceutical analysis and cover the use of reversed phase HPLC in the analysis of biomolecules.  A subsequent article will detail the application of HILIC, IEX, and SEC chromatography for the analysis if biomolecules. 

 

Amino Acids

Amino acids are made up of an amino group and a carboxylic acid moiety (Figure 1).  What is important is the difference in R group which gives the different amino acids (Table 1 and Figure 2).  There are only twenty naturally occurring amino acids in humans. 

Of the twenty natural amino acids eleven can be synthesized biologically from other ingredients while the remaining nine (the essential amino acids) must be consumed as part of a healthy diet.  These essential building blocks of life can be combined in a huge variety of ways to make up larger peptide and protein molecules.

Amino acids can be further split into four groups based on the functional groups within the amino acid structure; acidic, basic, neutral hydrophilic, and neutral hydrophobic (Table 1 and Figure 2).  

Figure 1: Generic amino acid structure (amino group highlighted in pink and carboxylic acid highlighted in green).

 
Acidic Basic Neutral, hydrophilic Neutral, hydrophobic
Aspartic acid
Glutamic acid
Arginine
Histidine
Lysine
Asparagine
Cysteine
Glutamine
Serine
Threonine
Tyrosine
Alanine
Glycine
Isoleucine
Leucine
Methionine
Phenylalanine
Proline
Tryptophan
Valine

Table 1: Amino acids.

 

Aspartic acid and glutamic acid have carboxy (weak) acid side chains.  They can also potentially be subjected to amidation side reactions rendering them neutral, hydrophilic.  There are three amino acids with basic side chains, compared to two with acidic side chains.  Furthermore arginine and, to a lesser extent lysine, are strongly basic. 

 
Non Polar
Polar
Electrically Charged - Acidic   Electrically Charged - Basic
 

Figure 2: Amino acid structures and properties.

 

When two or more amino acids bind together they form a peptide.  Highlighted in Figure 3 are the peptide bonds. 

 
Figure 3: Peptide (peptide bonds highlighted in red
 

Polypeptides are long, unbranched chains of amino acids which typically consist of 20-50 amino acid units.  Proteins consist of multiple polypeptides bound together.  It is easy to forget that proteins are not simply a strand of amino acids and that, if you have the amino acids assembled in the right order, you have the right product.

Those amino acid strands themselves form complex structures folding into sheets, helices, or simply as random chains.  These peptide units themselves are assembled into complex three dimensional structures, often reinforced by hydrogen bonding or more permanent disulfide bridges. 

However, these may also form dimers, trimers, tetramers or larger combinations.  Furthermore, it is estimated that 50% of our proteins are glycosylated and 2% of our genome codes for enzymes involved in glycosylation.

Proteins are vital to life and carry out many critical biological functions.  They do most of the work in cells and are required for the structure, function, and regulation of the body’s tissues and organs.  Proteins are generally categorized by their function within the human body.

  • Biochemical reactions - enzymes
  • Hormones - human growth hormone (hGH) regulates growth
  • Structural - collagen in bones, skin, hair etc.
  • Transport - O2 carried by hemoglobin in blood
  • Mechanical - actin in muscle movement
  • Defense/immune response - antibodies such as immunoglobulin G (IgG)

 

Pharmaceuticals vs. Biopharmaceuticals

Biopharmaceutical research is focused on the ability of proteins to act as defense and immune responders which is the basis of monoclonal antibody (mAb) studies.  Monoclonal means one type, therefore, each mAb is a copy of one type of antibody.  Monoclonal antibodies recognize and attach to specific proteins produced by cells.  Each monoclonal antibody recognizes one particular protein and work in different ways depending on the protein they are targeting. 

Traditional pharmaceutical research focusses on small molecules (i.e. aspirin) which are precisely defined entities that are chemically synthesized.  They are often stable compounds which have molecular weights of 100-1,500 Daltons and contain 1-5 reactive moieties.  In comparison biopharmaceutical compounds are genetically engineered from living cells and have molecular weights in the range 2,000-2,000,000 Da with 10-2,000 reactive groups. 

Biopharmaceuticals are often comprised of a mixture of closely related variants; therefore, there is a wider degree of variability (a degree of heterogeneity) in comparison with small molecule pharmaceuticals (Figure 4).  The combination of all of these factors make biopharmaceutical compounds incredibly complex and present unique analytical challenges in their separation and characterization.

 

Figure 4: Structure of aspirin vs. monoclonal antibody.

 

The wider degree of variability and heterogeneity exhibited by biopharmaceuticals does render them complex in terms of synthesis, purification, and characterization.  However, this trait also leads to the ability to produce compounds which mimic (biosimilars) or improve on (biobetters) the properties of the parent biopharmaceutical (i.e. improvements in efficacy, safety, drug disposition etc.).

The biopharmaceutical market place is not a new industry as it was established in the early 1980s (1982) with the introduction of insulin (a recombinant protein) into the pharmaceutical market place.  Whilst it is a mature market place there has been a surge in interest in biopharmaceutical compounds over the last few years, with particular interest being paid to protein biopharmaceuticals.  These types of compounds are produced using recombinant DNA technology and are used in the treatment of life threatening disease such as cancers and autoimmune diseases.  One feature which makes these types of compounds interesting as a therapeutic treatment is their ability to differentiate between healthy and unhealthy cells. 

The biopharmaceutical market now accounts for about 20% of the global pharmaceutical sales ($875 billion) with an expected increase to 50% of all new approved drugs being biopharmaceuticals by 2020 (Figure 5).

 

Figure 5: Proportion of the global pharmaceutical sales for traditional pharmaceutical and biopharmaceutical compounds.1

 

There are currently over 200 biopharmaceuticals on the market with an increasing number achieving blockbuster status (greater than $1bn sales per year, Table 2).  One quarter of the top 200 pharmaceutical compounds (by sales) in the USA in 2012 were biopharmaceuticals.2

 
Drug Brand Name Manufacturer Compound Class Therapeutic Area
Insulin Lantus Sanofi Protein (5 kDa) Diabetes
EPO Epogen/Aransep Amgen Glycoprotein (34 kDa) Blood anemia
Trastuzumab Herceptin Roche/Genentech mAb Breast cancer
Infliximab Remicade J&J mAb Arthritis, Crohn’s (AI)
Adalimumab Humira Abbvie mAb Arthritis, Crohn’s (AI)
Rituximab Mabthera Roche/Genentech mAb Lymphoma, leukemia, arthritis (AI)
Etanercept Enbrel Pfizer/Amgen Fusion protein Arthritis (AI)

Table 2: Examples of biopharmaceuticals currently on the market.

 

Biosimilars and Biobetters

A number of these biopharmaceutical drugs have come off or are due to come off patent which has led to increased activity in the development of biosimilars or biobetters.  Exact copies of the originator biopharmaceutical are impossible due to the complexity of these drugs. 

Biosimilars are approved versions of the originator drug that have been shown to have similar properties, efficacy, and safety.  A huge number of biosimilars have been approves in Asia, some of which possess a different primary protein sequence (amino acid order is different).  A number of biosimilars have also been approved in Europe; these are mainly therapeutic proteins (hGH and EPO) and the first mAb (Infliximab/Remicade) which was approved in 2013.  Currently there are no biosimilars on the market in the USA; however, new regulations are being implemented to facilitate the production of mAb biosimilars. 

Biobetters are not copies of the originator biopharmaceutical but are related to it by target or action.  Their properties are deliberately altered to enhance their efficacy, safety, or drug disposition.  Biobetters are generally produced by either an improvement in 1) the cell lines used to express the mAb or 2) the glycosylation profile of the mAb which may include genetic adaptation of the cell lines (the vast majority of biopharmaceuticals on the market are glycosylated).  An example of a biobetter which is in development by Glycotype GmbH is TrastuzuMab-GEX (TrasGEXTM) which incorporates an improved glycosylation profile over the originator. 

Table 3 shows biosimilars and biobetters which are currently in development. 

 
Biopharmaceutical Brand Sales ($bn) Biosimilars Biobetters
Humira Adalimumab $9.27 13 7
Remicade Infliximab $8.90 9 9
Enbrel Etanercept $7.87 21 8
Rituxan Rituximab $7.29 30 17
Herceptin Trastuzumab $6.40 24 12
Lantus Insulin glargine $6.40 5 2
Avastin Bevacizumab $6.26 14 9
Neulasta Pegfilgrastim $4.10 14 9
Lucentis Ranibizumab $3.72 2 2
Aransep Darbepoetin alfa $3.00 4 2
Epogen/Procrit Epoetin alfa $3.73 69 26
Novoseven Coagulation factor $1.50 8 12
Neupogen Filgrastim $1.44 52 17

Table 3: Biosimilars and biobetters currently in development.3

 

The process of manufacturing biopharmaceuticals is extremely important.  Chromatography is integral to several stages of the biopharmaceutical manufacturing process (Figure 6).  The production process is actually the biopharmaceutical product, not just the final compound, as it is the manufacturing process which determines the glycosylation, structure etc. of the biopharmaceutical which effects the efficacy, safety, and drug disposition.

 

Figure 6: Typical mAb manufacturing process.4

 

Biopharmaceutical Analysis

During development full characterization of the biopharmaceutical is required.  Due to their size, complexity, and heterogeneity analysis is typically more complex in comparison to the analysis of small molecules, therefore, typically a range of liquid chromatographic techniques alongside mass spectrometric detection are combined and utilized (Table 4).

 
Property Type of Chromatography
Charge Ion exchange chromatography (IEC), chromatofocusing, capillary zone electrophoresis (CZE), capillary isoelectric focusing(CIEF)
Size Size exclusion chromatography (SEC), CGE
Hydro(phob/phil)icity Reversed phase HPLC (RPLC), hydrophobic interaction chromatography (HIC), hydrophilic interaction chromatography (HILIC), micellar electrokinetic chromatography (MEKC)
Affinity Affinity chromatography

Table 4: Types of chromatography utilized in biopharmaceutical analysis (most highly used techniques shown in bold).

 

Amongst biopharmaceuticals monoclonal antibodies (mAb) are the most promising class of therapeutic molecules, therefore, the focus of this article will be primarily on the analysis of these types of molecules using IEC, SEC, RPLC, and HILIC.

The structure of a mAb is shown (Figure 7).  There are several characteristics inherent to mAbs which need to be considered to provide a detailed characterization of the compound. 

  • Amino acid sequence
  • Amino acid composition
  • Structural integrity
  • Higher order structures
  • Aggregation
  • S-S bridges
  • N- and O-glycosylation
  • N- and C-terminal sequence
  • Charge variants
  • Deamidation/isomerization
  • Oxidation
  • Clipping
 

Figure 7: Monoclonal antibody (mAb).

 

Many chemical modifications, which are generally unwanted, are introduced during the manufacturing process and storage and need to be characterized.
The characteristics of biopharmaceuticals can be determined at different levels (Figure 8). 

At the protein level the molecular weight (MW), structural integrity, charge variants, aggregation, and post-translational modifications can be determined.  Due to the size and complexity at the protein level it is often required to simplify the molecule through hydrolysis, digestion etc. to then analyze the smaller fragments. 

For example, acid hydrolysis of the protein will yield the individual amino acids which can be analyzed using HILIC to give the amino acid composition.  A very common strategy in biopharmaceutical analysis is known as peptide mapping in which the protein is digested (i.e. Trypsin digestion) to yield the corresponding peptides which are then analyzed to provide information on the amino acid sequence, modifications, modification sites, disulfide bridges etc. 

When using an appropriate enzyme, such as peptide-N-glycosidase (PNGase F), the sugar moieties can be cut from the protein and analyzed to determine the glycosylation profile of the protein.   

 

Figure 8: Biopharmaceutical characteristics determined at different levels. 

Reversed Phase Analysis - Proteins

Reversed phase HPLC (RPLC) analysis of proteins exhibits specific problems which would not normally be encountered when analyzing small molecules (Table 5).  Due to the very large number of positive charges on the protein molecule there are slow secondary interactions between the protein and the column surface which results in peak tailing. 

Peak tailing can be limited by using modern stationary phases which are designed to limit the molecules access to residual silanols on the silica surface, use ion-pairing reagents, i.e. trifluoroacetic acid (TFA), which will either pair with the molecule or the residual silanols and mitigate the effects of tailing, or use elevated temperatures up to 90 °C which can increase the kinetics of the secondary interactions and thus improve peak shape.

Peak broadening is also a common problem encountered during RPLC analysis of biopharmaceuticals and can be attributed to the low diffusion coefficient (Dm) and large size of biomolecules.  To limit band broadening efficient HPLC columns which are packed with sub-2 µm or core shell particles should be preferentially employed (Figure 9). 

When protein size is greater than 15 kDa it is very important to use a wide pore stationary phases (300 Å) should be used to limit band broadening.

 

Figure 9: Comparison of protein analysis on 5 µm and <2 µm particle columns.

 

Adsorption of proteins to the column surface or instrument components can also occur due to their strong hydrogen donor/acceptor properties and the numerous positive and negative charges within the protein.  Adsorption to the column can be overcome by using a less hydrophobic phase i.e. C4 instead of C18.  Adsorption of proteins to the instrument is being dealt with by many manufacturers through the production of BioInert systems. 

 
Problem Reason Solutions
Peak tailing
 
  • Slow secondary ionic interaction kinetics
  • Up to > 100 positive charges on proteins
  • Stationary phases with limited access to residual silanols
  • Ion-pairing reagent (TFA)
  • Higher temperature
Peak broadening
  • Low Dm of large molecules
  • Limited access to pores
  • Wide pore phases
  • Higher temperature
  • Efficient stationary phase
    (sub-2 µm particles, core-shell)
Adsorption
  • Strong hydrogen donor/acceptor
  • Numerous positive and negative charges
  • Less hydrophobic stationary phase
    (C4 vs. C18)
  • Dedicated LC system (BioInert)

Table 5: Problems encountered when analyzing biopharmaceuticals using reversed phase HPLC. 5

 

To simplify the analysis of mAbs it is possible to perform what is known as a middle-up analytical strategy in which the size and complexity of the protein is simplified by partial digestion using, for example, the enzyme papain which produces antibody fragments of ~50 kDa (Fab (fragment antibody) and Fc (fragment crystallizable) Figure 10). 

Alternatively the mAb disulfide bonds can be reduced using dithiothreitol (DTT) to produce fragments which are ~25 kDa (light chain (Lc)) and ~50 kDa (heavy chain (Hc)) in size.  The heterogeneity on the smaller fragments, in comparison to the intact mAb, is a lot easier to assess and these smaller fragments are much more amenable to analysis by chromatographic techniques such as RPLC.

 

Figure 10: Fragmentation of intact mAb.6

 

Figure 11 shows the RP analysis of Herceptin (which contains the mAb trastuzumab).  The chromatogram corresponding to the intact mAb shows a main peak for the main form of trastuzumab with some smaller, less well defined peaks at the base which correspond to other forms of trastuzumab which exist in the commercial solution.  These peaks demonstrate the large heterogeneity found in these large mAb compounds and can be attributed to charge or size variance or different glyco forms of trastuzumab.  Therefore, it can be seen that a mAb is never completely pure but contains other forms of the main compound. It should be noted that most mAb compounds are eluted with 30-40% organic modifier demonstrating their very similar hydrophobicity.  Therefore, a good generic starting mobile phase composition should contain 30-40% MeCN; this composition can then be optimized further if required.

Digestion of trastuzumab with papain produces the smaller ~50 kDa fragments Fab and Fc which can be separated under RPLC conditions (Figure 11).  Generally the Fc fragment is eluted first followed by the Fab fragment which exhibits a greater peak height and heterogeneity (which presents as additional small peaks at the base of the main Fc peak).  The amount of information which can be gained from this chromatogram over the chromatogram of the intact mAb is greatly increased due to the presence of many more peaks.

Finally, trastuzumab was reduced using DTT to produce light and heavy chain fragments (Lc ~25 kDa and Hc ~50 kDa respectively).  The RPLC chromatogram of the separation of Lc and Hc shows that the Hc fragment is more retained due to its larger size, furthermore, due to its larger size it exhibits more heterogeneity evidenced by the additional peaks within the chromatogram.  Mass spectrometry (MS) would be very useful to obtain information from the extra peaks within these chromatograms to give a full picture of the compounds present within the mAb solution.

To fully characterize a mAb the strategy outlined here in which the intact mAb and the fragments produced from digestion and reduction are analyzed is required to provide full characterization of these very complex molecules.     

 

Figure 11: Chromatogram of 1) intact mAb Herceptin (trastuzumab) 2) Fab and Fc fragments from papain digest 3) Lc and Hc fragments from dithiothreitol (DTT) reduction. Column: C8 100 x 2.1 mm, 1.8 µm, 300 Å.  Temperature: 80 °C.  Flow rate: 200 µL/min.  Mobile phase: A: 0.1% TFA, B: 0.1% TFA in MeCN, 30-38.6%B, 2-25 min.  Detector: UV 214 and 280 nm.  

 

All of the chromatograms shown in Figure 11 were obtained at a temperature of 80 °C - which could seem surprising due to the inherent instability of biomolecules. 

If trastuzumab or rituximab, for example, were analyzed in water at 80 °C they would be completely destroyed.  However, it can be shown that the analysis of mAbs can be improved under reversed phase conditions at elevated temperatures as the degradation kinetics and rate are different under RPLC conditions (Figure 12). 

The use of elevated mobile phase temperatures is advantageous and can impact adsorption, charge variant selectivity (tuning selectivity), peak widths, and retention.  In Figure 11 the chromatogram for intact rituximab shows no detectable peak at 40 or 50 °C, whereas, increasing the temperature to above 70 °C produces a well-defined peak.  The effect of temperature is less important and has a lesser impact on the chromatogram when working with fragments of mAbs. 

Analysis of reduced rituximab shows that the light chain fragment was poorly affected by temperature with little impact on the peak area with increasing mobile phase temperature.  Whereas, the heavy chain fragment was affected to a greater degree by increasing the temperature.

 

Figure 12: Effect of temperature on mAb analysis.7

 

Although there are several benefits to working at elevated temperatures the chromatogram should be monitored for the presence of ghost peaks which may indicate that there is on-column degradation which can be caused when working at high temperature and low pH (Figure 13).  At 90 °C analysis times of up to 15-20 minutes are acceptable for mAbs, beyond this, ghost peaks appear sporadically within the chromatogram which indicates that there is some on-column degradation which is probably related to instability of the mAb.

 

Figure 13: On-column mAb degradation.  Chromatogram showing no degradation (left) and chromatogram showing degradation (right).  Column: C18 150 x 2.1 mm, 3.6 µm core-shell.  Temperature 90 °C.  Detector: FLD 280-360 nm.  Injection volume: 0.5 µL.  Mobile phase: A: 0.05% TFA, B: 0.05% TFA in MeCN.  Left chromatogram 30-37%B, 6 min., 300 µL/min.  Right chromatogram 30-37%B, 60 min., 30 µL/min.  

 

Reversed Phase Analysis - Peptides

Another way to characterize large biomolecules is to digest the sample with trypsin to perform peptide mapping.  Trypsin cuts the protein at the lysine and arginine amino acids.  The final size of the peptide fragments range from 500 Da to 2 kDa, this smaller fragment size makes analysis by chromatographic techniques much more facile than analyzing the intact protein. 

The number of peptide fragments generated does need to be considered; typically 50-100 peptides will be produced for a mAb sample.  Therefore, the chromatographic technique and conditions employed need to have sufficient resolving power to differentiate between the peptide fragments.  To have high resolving power longer columns should be employed. 

The chromatogram in Figure 14 was obtained using a 250 x 2.1 mm column packed with 2.7 µm core-shell particles.  It can be seen that the large number of peptide fragments are resolved.  Addition of TFA helps to improve peak shape as it acts as an ion-pairing reagent by masking the positive charge at the peptide surface. 

However, it should be noted that TFA can cause problems with ionization efficiency in MS - although the benefits of the improved peak shape may outweigh any slight losses in MS sensitivity.   

 

Figure 14: Detailed reversed phase HPLC peptide map for Herceptin identity and purity assessment.  Column: C18, 2.1 x 250 mm, 2.7 µm core-shell.  Temperature: 60 °C.  Flow rate: 300 µL/min.  Detector: UV 214 nm.  Mobile phase: A: 0.05% TFA, B: 0.005% TFA in MeCN 1-45%B, 2-35 min.  Injection volume: 5 µL (2.4 µg).

 

A trypsin digest of trastuzumab (Figure 15) will generate 62 peptide fragments which must be differentiated chromatographically or using mass spectrometry to provide a full characterization; again illustrating the requirement for chromatographic techniques which provide high resolving capabilities. 

It can be seen from the chromatogram obtained from the trastuzumab tryptic digest that there are a vast number of peptide fragments which are resolved; however, there are a small number of hydrophilic peptides which are not retained under reversed phase HPLC conditions.  These hydrophilic peptides could be better retained using hydrophilic interaction chromatography (HILIC) to give a complete characterization of all the peptides produced from the tryptic digest. 

HILIC is being more commonly employed for peptide mapping applications due to its ability to retain and resolve hydrophilic peptides.  There are a few peptide fragments (highlighted in red in Figure 15) which are not part of the known peptide map for trastuzumab.  These peaks can be attributed to post-translational modifications such as deamidation, oxidation, glycosylation etc. 

 

Figure 15: Tryptic digest of trastuzumab to produce 62 peptides (top).  Peptide map of trastuzumab tryptic digest obtained under reversed phase HPLC conditions (bottom).

 

It can be seen from the chromatograms produced using reversed phase HPLC and MS that the applicability and high resolving power offered by this technique can provide immeasurable information when characterizing protein biopharmaceuticals.  

 

Typical Reversed Phase HPLC Conditions for Peptide Mapping

Detailed below are some general conditions which can be employed for RPLC peptide mapping.  UHPLC instrumentation and columns should be utilized to provide the high resolving power required to separate the large number of peptides produced from protein digestion.  The column should ideally be packed with core-shell particles and have a pore size in the region of 120-150 Å; larger pore sizes (i.e. 300 Å) are not required as the peptide fragments which are being analyzed are much smaller in size than the intact protein.  Smaller pore sizes ~80 Å are not suitable for peptide analysis. 

TFA at a reasonable concentration should be added to the mobile phase to improve peak shape.  Acetonitrile (MeCN) is the organic modifier of choice as it generates a reasonable pressure drop compared to methanol or other common organic solvents.  A 35 minute gradient is employed to give a good balance between resolving power and analysis time.  If the separation requires modification isopropanol (IPA) can be used to alter selectivity, however, the elution strength of IPA is greater than MeCN, and therefore, the gradient time should be adjusted appropriately.  Also the viscosity of IPA is higher than that of MeCN and will create a higher pressure drop across this system.

  • Instrument: UHPLC
  • Column: C18, 2.1 x 250 mm, < 3 µm, 120-150 Å core-shell particles
  • Mobile phase A: 0.05% TFA
  • Mobile phase B: 0.05% TFA in MeCN
  • Flow rate: 300 µL/min.
  • Gradient: 0-2 min: 1% B, 2-35 min: 1-45% B
  • Column temperature: 60 °C
  • UV detector: 214 and 280 nm
  • Acetonitrile is the organic modifier of choice
  • Isopropanol or 1-propanol can be used to change selectivity and optimize recovery (for proteins)

Reversed Phase Analysis - Amino Acids

When analyzing biomolecules the amino acid composition must be determined as it is critical in terms of regulatory submissions for both originator drugs and biosimilars.
Amino acids are very difficult to analyze under RPLC conditions in their native form due to their high polarity and low UV response.  Even if HILIC looked promising as a strategy for analysis there are currently no methods detailing analysis of the 20 amino acids without the use of mass spectrometry.  The best solution currently is to derivatize the amino acid using a UV or fluorotag such as OPA or FMOC and then analyze the derivatized materials using a C18 column and UV or fluorescence detection (Figure 16).  The following four step method (which can be automated) is widely employed for the analysis of amino acids.

  1. Hydrolysis to release the individual amino acids
  2. Labelling with a UV or fluorotag
  3. Chromatographic separation (typically C18)
  4. Detection and quantification

Two derivatization agents are commonly used (Figure 16).

  1. OPA (o-phthalaldehyde) for all primary amino acids
  2. FMOC (9-fluoroenylmethyl chloroformate) for secondary amino acids
 

Figure 16: OPA (o-phthalaldehyde) and FMOC (9-fluoroenylmethyl chloroformate) reaction with amines.

 

The chromatogram in Figure 17 shows the analysis of the amino acids in trastuzumab.  The chromatogram was obtained following the protocol for hydrolysis and derivatization which is described above.  Each of the amino acids can be easily identified and quantified using a standard calibration curve.

 

Figure 17: Reversed phase analysis of amino acid sequence of trastuzumab.

 

Summary

It can be seen that reversed phase HPLC is a very useful and powerful tool in the analysis of biopharmaceutical compounds.  Although there are several challenges which will be faced when analyzing these large complex molecules, the flexibility of modern RPLC (i.e. UHPLC instruments and columns etc.) provide adequate solutions to these challenges.  Any limitations of RPLC (i.e. retention of very hydrophilic peptides) can be solved through the utilization of other modes of chromatography such as HILIC, IEX, and SEC.  This wide toolkit of analytical techniques (combined with the power of MS) allows full characterization of biomolecules. 

 

References

  1. Sources: IMS Health, BCC Research, Pharmaceutical Technology, Biopharm. International, Reuters, PhRMA.
  2. Top 200 Pharmaceutical Products by US Retail Sales in 2012
  3. Rader, R. A. Bioprocess Intl. 2013 (June), 11.
  4. Carson, K. L., Nature Biotechnology 2005, 23, 1054-1058.
  5. Fekete, S.; Veuthey, J-. L.; Guillarme, D. J. Pharm. Biomed. Anal. 2012, 69, 9-27.
  6. Fekete, S.; Gassner, A-. L.; Rudaz, S.; Schappler, J.; Guillarme, G. Trends Anal. Chem. 2013, 42, 74-83.
  7. Fekete, S.; Rudaz, S.; Veuthey, J-. L.; Guillarme, D. J. Sep. Sci., 2012, 35, 3113-3123.
 

This article was based on our webcast
HPLC Techniques in Biopharmaceutical Analysis

You may also be interested in the following webcasts
Techniques Employed in Biopharmaceutical Analysis - Part I Reversed Phases and HILIC

 
 
 
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