Analyzing for Small Molecules in Large-Molecule Pharmaceuticals
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Wissenschaft und Technik
Originale
Analyzing for Small Molecules
in Large-Molecule Pharmaceuticals
The potential of capillary electrophoresis
Aldo Hörmann, Maria Schwarz, Fabian Stapf
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Solvias AG, Kaiseraugst, Switzerland
Zur Verwendung mit freundlicher Genehmigung des Verlages / For use with permission of the publisher l
Corresponding author: Dr. Aldo Hörmann, Solvias AG, Römerpark 2,
4303 Kaiseraugst, Switzerland, e-mail: aldo.hoermann@solvias.com
■ ■ AB ST R AC T 1. Introduction
All biopharmaceuticals contain small molecules. Some are Biopharmaceuticals contain large molecules as active in
introduced during upstream manufacture of the biophar- gredients but still small molecules are present in all bio
maceutical and must be removed later during downstream pharmaceuticals. For example, in upstream manufacture
processing. Still others are added in the formulation to get of recombinant proteins, small molecules are needed as
a stable drug product. Analysis of biopharmaceuticals for nutrients of the protein producing organisms. Others are
small molecules is thus critical to ensure safety and efficacy used in downstream processing, e. g. reducing agents to
and for process development. Capillary electrophoresis (CE) cleave disulfide bonds. Formulations of biopharmaceuti
is a very powerful method to assess small molecules. In this
cals contain small molecule buffers and other ingredients
article we review the technology and illustrate the versatil-
ity and potential of the method to a number of analytes such as surfactants to improve stability of the protein.
in samples typically obtained in the production and final Safety and efficacy of biopharmaceuticals depend on effi
release of biopharmaceuticals. Recently, we worked suc- cient removal of undesired substances in the downstream
cessfully on quantitation of cyclodextrin in a formulation by process and the correct amount of necessary ingredients
CE. We will present the results of this exploratory study on in the final formulation. It is thus of utmost importance
cyclodextrins which are becoming an ever more important that sensitive and specific analytical methods are avail
additive in protein formulations. able to monitor the critical small molecules in the final
drug product or in in-process samples. Such methods
and related instrumentation should be sufficiently robust
■ ■ ZU S AMM E N FA S SU N G
such that they can be used in a routine quality control
Analytik von kleinen Molekülen in Biopharmazeu- (QC) environment. Often the methods must be validated
tika / Das Potential der Kapillarelektrophorese following GMP requirements.
Alle Biopharmazeutika enthalten kleine Moleküle. Diese Obviously, a big arsenal of analytical methods exists to
werden beispielsweise im Upstream des Produktionsprozes- quantify small molecules in protein solutions. The work
ses eingebracht und müssen später im Downstream entfernt horse is certainly HPLC with UV detection which can cover
werden. Ferner werden kleine Moleküle gezielt Formulie- the needs of many applications. The method is, however, not
rungen zugegeben, um stabile Arzneimittel zu erhalten. Die suitable for analytes which carry no chromophores such as
Analytik von Biopharmazeutika hinsichtlich kleiner Molekü- small ions, buffers, reducing agents, etc. In such cases other
le ist somit eine entscheidende Voraussetzung zur Gewähr- detection methods can be used such as refractive index
leistung der Patientensicherheit und der Wirksamkeit sowie
(RI) or evaporative light scattering (ELS). Alternatively ion
für die Prozessentwicklung. Kapillarelektrophorese (CE) ist
eine leistungsfähige Methode zur Bestimmung von kleinen exchange chromatography is frequently used. For volatile
Molekülen. In diesem Beitrag wird die Technologie erläutert analytes gas chromatography is a frequently used method,
und ihre breite Verwendbarkeit und ihr Potential anhand usually with flame ionisation detection (FID).
einiger Analyten aufgezeigt, die typischerweise in der Pro- These more common
duktion und der Freigabe von Biopharmazeutika bestimmt ■ ■ K EY W O R D S methods obviously have
werden müssen. Cyclodextrine werden immer wichtiger als • Biopharmaceuticals their justifications and
Zusatz in Proteinformulierungen. Kürzlich konnten mittels • Capillary electrophoresis clear advantages, else
CE erfolgreich Cyclodextrine in Formulierungen analysiert • Small molecules they would not be used
werden; die Resultate dieser exploratorischen Studie werden that frequently. However,
hier vorgestellt. Pharm. Ind. 73, Nr. 12, 2222 – 2228 (2011) there are cases where the
Pharm. Ind. 73, Nr. 12, 2222 – 2228 (2011)
© ECV ∙ Editio Cantor Verlag, Aulendorf (Germany) Hörmann et al. ∙ Capillary electrophoresis 1
Wissenschaft und Technik
Originale
workhorse methods do not perform as desired, for exam filled with an electrolyte solution [1 – 3]. Positively charged
ple due to difficulties with the matrix, lack of chromo ions are attracted by the cathode and vice versa. The speed
phores, insufficient selectivity or sensitivity. In this paper at which an ion migrates depends on the net charge of the
we focus on capillary electrophoresis (CE) as an efficient ion and its hydrodynamic size. A somewhat special effect
and robust tool for the analysis of many small molecules of CE is the so-called electroendosmotic flow (EOF). As the
typically encountered in biopharmaceuticals. For long, inside of the fused-silica capillary is negatively charged,
CE has been a somewhat exotic method that was not al the liquid inside the capillary has an overall positive
ways well received in typical QC labs as it was considered charge (Fig. 2). Therefore the whole inside of the capillary
a difficult method to be run by experts only. With the ad travels towards the cathode. Consequently, even negative
vent of biopharmaceuticals and improved instrumenta ly charged ions are carried with the EOF towards the cath
tion, however, this has changed. For biopharmaceuticals ode, unless their migration speed is faster than the EOF.
Zur Verwendung mit freundlicher Genehmigung des Verlages / For use with permission of the publisher l
CE is quite commonly used in QC of the protein and is The most common use of CE is “capillary zone electro
more and more replacing previously used classical gel- phoresis” (CZE) where a mixture of analytes is introduced
based electrophoretic methods such as SDS-PAGE and as a small zone into the capillary by briefly dipping the cap
isoelectric focusing (IEF). A CE instrument can now be illary into the solution to be analyzed and applying some
found in most biopharma QC laboratories and can thus pressure. Separation of the analytes starts by turning on the
be used for small molecule analysis as well. high voltage. Another application of CE is capillary isoelec
tric focusing (cIEF) which is typically used in the analysis of
proteins. In this technique substances with multiple ioniza
2. Brief review of CE technology
tion centres travel in a capillary filled with a medium that
Capillary electrophoresis in general is based on applying has a pH gradient. Substances travel in this gradient until
a high voltage (some 5 – 30 kV) across a capillary (Fig. 1) they reach a pH at which they carry no net charge (isoelec
Fig. 1: Schematic representation of capillary electrophoresis with UV detection. The capillary can be switched between the buffer
and the sample.
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Fig. 2: Origin of the electroendosmotic flow. Negative charges are generated at the capillary wall, e. g. due to ionization of silanol
groups. This results in a net positive charge of capillary content, in particular near the capillary wall, which is attracted by the
cathode and thus causing the flow.
Pharm. Ind. 73, Nr. 12, 2222 – 2228 (2011)
2 Hörmann et al. ∙ Capillary electrophoresis © ECV ∙ Editio Cantor Verlag, Aulendorf (Germany)
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Zur Verwendung mit freundlicher Genehmigung des Verlages / For use with permission of the publisher l
Fig. 3: Principle of indirect UV detection. The background electrolyte absorbs the light from the light source until a non-absorbing
analyte passes by creating a “hole” which allows the light to reach the detector. This event is detected as a negative signal on the
absorbance scale.
tric point). This means that separation in this technique is this setting, the detector always sees an absorption of UV
based solely on the isoelectric point of each substance. In light. A non-absorbing analyte passing by the detector
this paper, however, we will focus on CZE. will reduce the absorption, basically creating a “hole” in
Detection of the analytes can be achieved by several the electrolyte. The detector therefore registers a negative
techniques, including UV, laser induced fluorescence (LIF), signal whenever an analyte passes by. This technology has
contactless conductivity or even by mass spectrometry, been proven to be very useful for the detection of many
the latter still being a technique reserved to research and non-UV-active ions relevant to biopharmaceuticals and we
development. LIF detection is highly sensitive and is thus will show a number of examples in the following chapters.
very useful for trace analysis. However, the analytes need
to have a fluorophore themselves or they must be linked to
3. Analysis of typical ions in large molecule
a fluorophore before analysis which can be tricky. In this
pharmaceuticals
paper we will focus on detection by UV. For direct detec
tion, the analyte must carry a chromophore such that light Analytical methods used for in-process controls or final
is absorbed at a useful wavelength. However, especially release of biopharmaceuticals must be robust and run on
small ions often do not have a suitable chromophore and common instrumentation. The methods should at least be
therefore cannot be detected by UV. In this case, so-called qualified and for final release fully validated according to
indirect detection is useful. As shown in Fig. 3, the capil ICH Q2 guidelines. In our labs we have recently successfully
lary is filled with an electrolyte that absorbs UV light. In developed a number of CZE methods for ions in biophar
■ ■ Table 1
Some CZE methods with indirect UV detection for the analysis of small ions in recombinant protein phar-
maceuticals. LOQ: limit of quantitation. IPC: in-process control.
Analyte LOQ Used for Status
TCEP (tris(2-carboxyethyl)phosphine) including oxidized TCEP 10 ppm final release validated
TRIS (tris(hydroxymethyl)aminomethane) 10 ppm IPC validated
Histidine 10 ppm IPC validated
TFA (trifluoroacetate) 5 ppm final release validated
Citrate 5 ppm IPC qualified
Guanidine 5 ppm IPC qualified
Magnesium 5 ppm IPC qualified
Potassium 5 ppm IPC qualified
Muramic acid 0.45 % IPC qualified
N-Acetyl-muramic acid 250 ppm IPC qualified
Pharm. Ind. 73, Nr. 12, 2222 – 2228 (2011)
© ECV ∙ Editio Cantor Verlag, Aulendorf (Germany) Hörmann et al. ∙ Capillary electrophoresis 3
Wissenschaft und Technik
Originale
■ ■ Table 2 es. Methods are typically based on commercially available
kits from Beckman and MicroSolv/Analis but required sig
Accuracy of TCEP determination by CZE in PEG nificant development to obtain a stable method.
ylated protein as measured by recovery of the As a case study some more details on the method for
TCEP (tris(2-carboxyethyl)phosphine) and oxidized TCEP
indicated spike. The protein was presented in two are reported here. TCEP is used as an alternative to DTT
different buffers. (dithiothreitol) in order to rupture disulfide bonds during
downstream processing. The goal of the analytical method
Buffer 1 Buffer 2
was to detect traces of residual TCEP and its oxidized deriv
Spike level (ppm) Recovery (%) Spike level (ppm) Recovery (%) ative in the final drug product, a PEGylated protein which
10 126 10 74 % is a rather difficult matrix. The method uses a CElixirOA 5.4
Zur Verwendung mit freundlicher Genehmigung des Verlages / For use with permission of the publisher l
50 90 50 91 % kit from MicroSolv and runs on a standard Beckman PA800
100 83 100 107 % instrument equipped with a 60-cm fused silica capillary.
Fig. 4 shows a typical electropherogram. The runtime of the
method is just 5 min allowing for rapid analysis which is
maceuticals (Table 1). Clearly, CZE is a powerful method important in particular during development of a process
to analyze for a large variety of different ions with typically and stability studies. Due to the complexity of the matrix,
excellent sensitivity. All methods are qualified and some the baseline is not flat but highly reproducible as shown by
of the methods were fully validated. The main reason to an excellent repeatability with a relative standard devia
develop these methods on CZE was that HPLC-UV was not tion of just 3 % at 10 ppm TCEP. Good linearity (Fig. 5) and
suitable due to the lack of a good chromophore. For the accuracy (Table 2) were found, too. This data demonstrates
same reason, indirect UV detection was chosen in all cas the power of the method as a routine QC method.
Fig. 4: Typical electropherogram of a test solution of a PEGylated protein containing TCEP (10 ppm) and oxidized TCEP. Peaks
were inverted due to indirect UV detection.
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Fig. 5: Linearity of the method to analyze TCEP in PEGylated protein.
Pharm. Ind. 73, Nr. 12, 2222 – 2228 (2011)
4 Hörmann et al. ∙ Capillary electrophoresis © ECV ∙ Editio Cantor Verlag, Aulendorf (Germany)
4. Cyclodextrins in biopharmaceutical Hydroxypropyl-β-cyclodextrin (HP-CD) has been shown
formulations to inhibit agitation-induced aggregation of antibodies [5].
Analytical methods are needed to determine the content
Cyclodextrins (CDs) have great potential in inhibiting of CDs in protein formulations. The analytical task is not
aggregation of proteins [4]. In particular for monoclonal straight-forward as CDs lack a chromophore and can at
antibodies that are formulated at ever increasing concen tach non-covalently to the protein. To our knowledge no
trations, prevention of aggregation is crucial. Thus the use standard analytical method is available to quantify CDs
of CDs as formulation ingredients is heavily investigated. in protein formulations.
CDs are not charged and thus are not directly suited
for analysis by CE. However, CDs can undergo complexa
tion of organic acids and thus become charged. Actually,
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Zur Verwendung mit freundlicher Genehmigung des Verlages / For use with permission of the publisher l
we use the concept of complexation by a complexing
agent (CA) to achieve three goals: First, the CD gains a
charge and thus can be sepearted by CZE. Second, com
plexation with the CA is competing with complexation of
the protein (Fig. 6), so if a sufficiently high concentration
Fig. 6: Complexation of CD with a protein in a protein formula- of CA is used, the CD is freed from the protein and can be
tion and competing complexation with a complexing agent
(CA). For analytical purposes the equilibrium should be on analyzed. Third, if a suitable CA with a chromophore is
the side of the CD (CA) complex which is true under the stated used as complexing agent, it can be used as a background
requirement. UV absorber for indirect CZE.
Fig. 7: Analysis of HP-CD (≈ 10 %) in a 10 mg/mL protein solution. Indirect detection at 254 nm with IBA in the background
electrolyte, pH = 8.
■ ■ Table 3
Summary of results to check the validity of CZE method to analyze HP-CD in protein solution and placebo
formulation. RSD: relative standard deviation.
Parameter Quantity Protein solution Placebo
Repeatability at 1 % RSD (n = 6)Wissenschaft und Technik
Originale
Zur Verwendung mit freundlicher Genehmigung des Verlages / For use with permission of the publisher l
Fig. 8: Analysis of different CDs by CZE with IBA as complexing agent and background electrolyte, pH = 8.
We chose to use 3-iodo-benzoic acid (IBA) as a com small ions to cyclodextrins in biopharmaceuticals in a rou
plexing agent to analyze HP-CD in a protein solution tine QC setting. While other methods, in particular HPLC
by CZE with indirect UV detection at 254 nm. A typical methods, will remain the workhorses in QC, capillary elec
electropherogram is shown in Fig. 7. The method was trophoresis will gain further importance as an additional
successfully checked for typical validation parameters rapid method for otherwise difficult analytical tasks.
critical for routine analysis (Table 3).
To check the power of this CZE analytical approach to
CDs, we have investigated different HP-CD from differ
ent vendors and compared it to non-hydroxypropylated
β-cyclodextrin. As shown in Fig. 8, the HP-CD from Beck
man and Aldrich show a very similar electropherogram, as ■■REF ERENC E S
expected. The β-cyclodextrin results in a much sharper peak
than HP-CD further illustrating the specificity of the method. [1] Foret F, Křivánková L, Boček P. Capillary zone electrophoresis.
Weinheim: VCH Verlagsgesellschaft mbH; 1993.
Overall CZE is a powerful method to analyze protein [2] Engelhardt H, Beck W, Schmitt T. Kapillarelektrophorese. Braun
solutions for CDs with great potential in development schweig: Vieweg; 1994.
and routine analysis. [3] Agilent Technologies. High performance capillary electrophoresis,
a primer. 2009.
[4] Serno T, Geidobler R, Winter G. Protein stabilization by cyclo
dextrins in the liquid and dried state. Adv Drug Deliv Rev. 2011. In
5. Conclusion press.
[5] Serno T, Carpenter JF, Randolph TW, Winter GJ. Inhibition of
In this paper we have demonstrated the versatility of CZE agitation-induced aggregation of an IgG antibody by hydroxypro
as a tool to reliably quantify small molecules ranging from pyl-beta-cyclodextrin. J Pharm Sci. 2010;99 : 1193 – 1206.
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