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**Chapter 8**

**Novel Analytical Tools**

Christian W. Huck

**1. Introduction**

of analysis.

http://dx.doi.org/10.5772/51915

**for Quality Control in Food Science**

Additional information is available at the end of the chapter

Due to the fast technological and data treatment advancements new insights into food can be considered. The application of these novel analytical techniques belongs to the responsi‐ bility of food chemists and analysts. Thereby, an increase in efficiency is based on an im‐ proved lower limit of detection (LOD), selectivity to separate analytes of interest and speed

High-performance liquid chromatography (HPLC) belongs to the traditional separation techniques applied to a broad range of hydrophilic and hydrophobic ingredients in both the reversed-phase (RP) [1] as well as normal-phase (NP) [2] mode. In a conventional HPLC system the inner diameter of the separation column, which is the core of the sepa‐ ration unit, is 4.6 mm. During the last decade miniaturization down to 20 μm allowed to increase on one side the sensitivity and on the other side speed of analysis could be en‐ hanced dramatically. Therefore, novel stationary phases mainly based on polymers have been designed and brought to the market to enable both the separation of low and highmolecular weight analytes [3]. As an alternative separation technique capillary electropho‐ resis (CE), which separates analytes due to their different ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm can be applied [4]. Thereby, the appearance of the electroosmotic flow (EOF) can influence the separation efficiency by either speeding up the separation process or by improving the resolution. Capillary electrochromatography (CEC) is a hybrid technique of both HPLC and CE in which both pressure and an electri‐ cal field are applied and enables extreme high resolution. The drawback of this separa‐ tion method is the fact, that real samples can hardly be analysed due to the disturbance by the matrix [5]. In many cases the analyte of interest is only available in very low con‐

> © 2012 Huck; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Huck; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

#### **Chapter 8**

## **Novel Analytical Tools for Quality Control in Food Science**

Christian W. Huck

[120] St, Angelo. A. J., Crippen, K. L., Dupuy, H. P., & James Jr, C., Jr. (1990). Chemical and sensory studies of antioxidant-treated beef. *Journal of Food Science*, 55, 1501-1539.

[121] James, J. M., & Calkins, C. R. (2005). Cooking rate and holding time effect on the fla‐ vor of muscles from the beef chuck and the round. In Proceedings of the internation‐

[122] Elmore, J. S. (2008). Aroma. In Nollet L. M. L and Toldra. F (eds.) Handbook of mus‐

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[124] Moon, S. Y., Cliff, M. A., & Li-Chan, E. C. Y. (2006). Odour-active components of si‐ mulated beef flavor analyzed by solid phase microextraction and gas chromatogra‐ phy-mass spectrometry and-olfactometry. *Food Research International*, 39, 294-308.

[125] Xie, J. C., Sun, B. G., & Wang, S. B. (2008). Aromatic constituents from Chinese tradi‐ tional smoke-cured bacon of mini-pig. *Food science and technology international*, 14,

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cle foods analysis Boca Raton CRC , 242-262.

1081-1087.

176 Latest Research into Quality Control

329-340.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51915

#### **1. Introduction**

Due to the fast technological and data treatment advancements new insights into food can be considered. The application of these novel analytical techniques belongs to the responsi‐ bility of food chemists and analysts. Thereby, an increase in efficiency is based on an im‐ proved lower limit of detection (LOD), selectivity to separate analytes of interest and speed of analysis.

High-performance liquid chromatography (HPLC) belongs to the traditional separation techniques applied to a broad range of hydrophilic and hydrophobic ingredients in both the reversed-phase (RP) [1] as well as normal-phase (NP) [2] mode. In a conventional HPLC system the inner diameter of the separation column, which is the core of the sepa‐ ration unit, is 4.6 mm. During the last decade miniaturization down to 20 μm allowed to increase on one side the sensitivity and on the other side speed of analysis could be en‐ hanced dramatically. Therefore, novel stationary phases mainly based on polymers have been designed and brought to the market to enable both the separation of low and highmolecular weight analytes [3]. As an alternative separation technique capillary electropho‐ resis (CE), which separates analytes due to their different ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm can be applied [4]. Thereby, the appearance of the electroosmotic flow (EOF) can influence the separation efficiency by either speeding up the separation process or by improving the resolution. Capillary electrochromatography (CEC) is a hybrid technique of both HPLC and CE in which both pressure and an electri‐ cal field are applied and enables extreme high resolution. The drawback of this separa‐ tion method is the fact, that real samples can hardly be analysed due to the disturbance by the matrix [5]. In many cases the analyte of interest is only available in very low con‐

© 2012 Huck; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Huck; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

centrations. Therefore, selective enrichment and purification steps are the method of choice, which can be accomplished by solid-phase extraction (SPE). Therefore, a material designed for a special analytical question is filled into a cartridge or pipette tip and the sample of interest is put onto the material in liquid form [6]. In the following, analytes of interest can interact with the functional groups of the stationary phase and compounds being not of interest can simply be washed away. In the final elution step, only some mi‐ cro liters of liquid are required to elute the analytes of interest from the stationary phase being available in relatively high concentrations for the following analytical steps. The fol‐ lowing analytical procedure can be either a separation or spectroscopic method. Spectro‐ scopic methods at this stage of the analytical procedure either include mass spectrometry (MS) and/or vibrational spectroscopy, respectively. In MS most of the samples are ana‐ lysed applying electrospray ionization (ESI) as an interface with different types of mass detectors including e.g. time of flight (TOF), ion trap, ion cyclotron and quadrupoles. As an alternative, matrix assisted laser desorption ionization time of flight mass spectrome‐ try (MALDI-TOF/MS) can be applied for the determination of high molecular weight com‐ pounds including proteins, peptides and lipids. For the analysis of low molecular ingredients < 1000 Da the so called matrix-free laser desorption ionization (mf-LDI) MS technique must be applied [7].Vibrational spectroscopy in the field of food analysis is mainly applied in the mid (400 – 4000 cm-1) as well as in the near infrared (4000 – 12000 cm-1) of the electromagnetic spectrum. In combination with chemo metrical algorithms these methods can be used for the authentication of the material on one hand, on the oth‐ er hand quantitative analysis allows to control selected quality parameters [8].

detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the spe‐ cific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical han‐ dling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the mid‐ dle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to be ana‐ lysed is carried out with only a few microliters causing a dramatic increase in concen‐ tration from which further analytical investigations can benefit due to the easier

Novel Analytical Tools for Quality Control in Food Science

http://dx.doi.org/10.5772/51915

179

4 *Figure 1. Multidimensional analytical approach*

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of surface to size, which results in a high capacity and allows analyte detection with high sensitivity down to the femtomole range in the case when mass spectrometry is applied for detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the specific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical handling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the middle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to

6 *Sample enrichment/purification*

3

handling of the systematic investigation.

1 2 3

5

**Figure 1.** Multidimensional analytical approach

Running Title

In the following a systematic analytical approach is introduced, which allows combining the different analytical techniques in a synergistic manner to get deeper insights into the compo‐ sition and origin of food samples.

#### **2. Systematic analytical approach**

The key technologies described in the above chapter can be combined according to the scheme depicted in Figure 1. In this approach extraction of the material for the further ana‐ lytical steps and individual procedures can be linked to sample enrichment/purification, separation, vibrational spectroscopy and mass spectroscopy followed by database analysis. The different parts are described in the following sub-chapters.

#### **2.1. Sample enrichment/purification**

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of sur‐ face to size, which results in a high capacity and allows analyte detection with high sen‐ sitivity down to the femtomole range in the case when mass spectrometry is applied for

3

detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the spe‐ cific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical han‐ dling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the mid‐ dle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to be ana‐ lysed is carried out with only a few microliters causing a dramatic increase in concen‐ tration from which further analytical investigations can benefit due to the easier handling of the systematic investigation.

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of surface to size, which results in a high capacity and allows analyte detection with high sensitivity down to the femtomole range in the case when mass spectrometry is applied for detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the specific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical handling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the middle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to

4 *Figure 1. Multidimensional analytical approach*  **Figure 1.** Multidimensional analytical approach

6 *Sample enrichment/purification*

2 3

5

Running Title

centrations. Therefore, selective enrichment and purification steps are the method of choice, which can be accomplished by solid-phase extraction (SPE). Therefore, a material designed for a special analytical question is filled into a cartridge or pipette tip and the sample of interest is put onto the material in liquid form [6]. In the following, analytes of interest can interact with the functional groups of the stationary phase and compounds being not of interest can simply be washed away. In the final elution step, only some mi‐ cro liters of liquid are required to elute the analytes of interest from the stationary phase being available in relatively high concentrations for the following analytical steps. The fol‐ lowing analytical procedure can be either a separation or spectroscopic method. Spectro‐ scopic methods at this stage of the analytical procedure either include mass spectrometry (MS) and/or vibrational spectroscopy, respectively. In MS most of the samples are ana‐ lysed applying electrospray ionization (ESI) as an interface with different types of mass detectors including e.g. time of flight (TOF), ion trap, ion cyclotron and quadrupoles. As an alternative, matrix assisted laser desorption ionization time of flight mass spectrome‐ try (MALDI-TOF/MS) can be applied for the determination of high molecular weight com‐ pounds including proteins, peptides and lipids. For the analysis of low molecular ingredients < 1000 Da the so called matrix-free laser desorption ionization (mf-LDI) MS technique must be applied [7].Vibrational spectroscopy in the field of food analysis is mainly applied in the mid (400 – 4000 cm-1) as well as in the near infrared (4000 – 12000 cm-1) of the electromagnetic spectrum. In combination with chemo metrical algorithms these methods can be used for the authentication of the material on one hand, on the oth‐

er hand quantitative analysis allows to control selected quality parameters [8].

sition and origin of food samples.

178 Latest Research into Quality Control

**2. Systematic analytical approach**

**2.1. Sample enrichment/purification**

The different parts are described in the following sub-chapters.

In the following a systematic analytical approach is introduced, which allows combining the different analytical techniques in a synergistic manner to get deeper insights into the compo‐

The key technologies described in the above chapter can be combined according to the scheme depicted in Figure 1. In this approach extraction of the material for the further ana‐ lytical steps and individual procedures can be linked to sample enrichment/purification, separation, vibrational spectroscopy and mass spectroscopy followed by database analysis.

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of sur‐ face to size, which results in a high capacity and allows analyte detection with high sen‐ sitivity down to the femtomole range in the case when mass spectrometry is applied for

**Figure 2.** TiO2, ZrO2 incorporated into a polymer matrix for phosphopeptide enrichment

Immobilisation of such polymers into pipette tips with trypsin can be used for fast digestion of peptides and proteins within only a few minutes ensuring high capacity and sequence coverage (Figure 3) even in the high-throughput mode using robotic pipetting systems [10]. In comparison to this quite young approach the conventional digestion procedure lasts ap‐ proximately 24 hours and doesn´t show in any case better results by higher sequence cover‐ ages. For this reason this approach is of high interest for the routine analysis and/or diagnostics, respectively. As a carrier glycidylmethacrylate-co-divinylbenzene (GMA/DVB) polymerized in pipette tips was chosen. The major advantages of in-tip digestion are easy handling and small sample amount required for analysis. Microwave-assisted digestion was applied for highly efficient and time saving proteolysis. Adaption to an automated robotic system allowed fast and reproducible sample treatment. Investigations with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography coupled to electrospray-ionization mass spectrometry (LC-ESI/MS) attest‐ ed high sequence coverages (SCs) for the three standard proteins, myoglobin (Myo, 89%), bovine serum albumin (BSA, 78%) and alpha-casein (α-Cas, 83%). Compared to commercial‐ ly available trypsin tips clear predominance concerning the digestion performance was ach‐ ieved. Storability was tested over a period of several weeks and results showed only less decrease (<5%) of protein sequence coverages. The application of microwave-assisted in-tip digestion (2 minutes) with full automation by a robotic system allows high-throughput anal‐ ysis (96 samples within 80 minutes) and highly effective proteolysis.

**Figure 3.** Trypsin immobilized pipette tips for high-throughput analysis of peptides

Material enhanced laser desorption ionisation (MELDI) is a method, which is based on the conventional matrix assisted laser desorption ionisation time of flight mass spectrometric (MALDI-TOF/MS) detection with the significant difference that before LDI MS step a selec‐ tive enrichment procedure is carried out for the distinct analysis of a certain compound class. Compared to other similar techniques in this field, this approach benefits from the physical properties of the material itself (pore size, surface area, capacity, etc.) and its chemi‐ cal derivatisation/functionalisation. In the past this technique was proven to be highly effi‐ cient for the analysis of biomarkers following an optimised strategy (Figure 4). In the first step a selected material including e.g., nanotubes, fullerenes, nano-crystalline diamond, pol‐ ymers, cellulose, etc., which are derivatised with functional groups (C18, IMAC (immobi‐ lised metal affinity chromatography), IEX and others) is activated and the serum sample of interest is incubated. During this step, selective binding of molecules according to their functional group is achieved and finally undesired components can be washed away apply‐ ing an optimised protocol. In the next step the incubated material is put onto a conventional

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**2.2. Selective MELDI-carriers**

**Figure 3.** Trypsin immobilized pipette tips for high-throughput analysis of peptides

#### **2.2. Selective MELDI-carriers**

**Figure 2.** TiO2, ZrO2 incorporated into a polymer matrix for phosphopeptide enrichment

180 Latest Research into Quality Control

ysis (96 samples within 80 minutes) and highly effective proteolysis.

Immobilisation of such polymers into pipette tips with trypsin can be used for fast digestion of peptides and proteins within only a few minutes ensuring high capacity and sequence coverage (Figure 3) even in the high-throughput mode using robotic pipetting systems [10]. In comparison to this quite young approach the conventional digestion procedure lasts ap‐ proximately 24 hours and doesn´t show in any case better results by higher sequence cover‐ ages. For this reason this approach is of high interest for the routine analysis and/or diagnostics, respectively. As a carrier glycidylmethacrylate-co-divinylbenzene (GMA/DVB) polymerized in pipette tips was chosen. The major advantages of in-tip digestion are easy handling and small sample amount required for analysis. Microwave-assisted digestion was applied for highly efficient and time saving proteolysis. Adaption to an automated robotic system allowed fast and reproducible sample treatment. Investigations with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography coupled to electrospray-ionization mass spectrometry (LC-ESI/MS) attest‐ ed high sequence coverages (SCs) for the three standard proteins, myoglobin (Myo, 89%), bovine serum albumin (BSA, 78%) and alpha-casein (α-Cas, 83%). Compared to commercial‐ ly available trypsin tips clear predominance concerning the digestion performance was ach‐ ieved. Storability was tested over a period of several weeks and results showed only less decrease (<5%) of protein sequence coverages. The application of microwave-assisted in-tip digestion (2 minutes) with full automation by a robotic system allows high-throughput anal‐

Material enhanced laser desorption ionisation (MELDI) is a method, which is based on the conventional matrix assisted laser desorption ionisation time of flight mass spectrometric (MALDI-TOF/MS) detection with the significant difference that before LDI MS step a selec‐ tive enrichment procedure is carried out for the distinct analysis of a certain compound class. Compared to other similar techniques in this field, this approach benefits from the physical properties of the material itself (pore size, surface area, capacity, etc.) and its chemi‐ cal derivatisation/functionalisation. In the past this technique was proven to be highly effi‐ cient for the analysis of biomarkers following an optimised strategy (Figure 4). In the first step a selected material including e.g., nanotubes, fullerenes, nano-crystalline diamond, pol‐ ymers, cellulose, etc., which are derivatised with functional groups (C18, IMAC (immobi‐ lised metal affinity chromatography), IEX and others) is activated and the serum sample of interest is incubated. During this step, selective binding of molecules according to their functional group is achieved and finally undesired components can be washed away apply‐ ing an optimised protocol. In the next step the incubated material is put onto a conventional steel target used in MALDI-TOF/MS, a matrix substance is added (e.g., sinapinic acid) and finally the mass spectrum is generated by the laser desorption ionisation process. The result is a mass spectrum being characteristic for a patient and/or the nutrition profile. Multivari‐ ate analysis (MVA) can be applied for further data analysis and interpretation, a clustering into certain stages of an illness can be achieved, respectively. From the mass spectrum po‐ tent biomarker molecules can be selected and identified by further analytical steps. The bio‐ marker itself and/or the profile of the corresponding mass spectrum can be used for the screening of certain diseases, stages therefrom, allergies, nutrition effects and so on [7].

rosity can be adjusted [12]. Extensive investigations on polymerisation time and tempera‐ ture have been carried out enabling a tailored design of micro-, meso- and macro-pore distribution [13, 14]. This results in the applicability of such capillaries with an inner diame‐ ter between 20 and 200 μm for even the separation of high- and low-molecular weight com‐ pounds. These capillaries can be highly successfully applied analysing peptides, proteins, oligonucleotides, DNA fragments as well as "small molecules" such as phenols, flavonoids, catechins, acids etc. Figure 5 shows as an example the separation of olive oil ingredients. This separation is characterised by a very high ratio of flow to back pressure, which is of high interest to perform extremely rapid Coupling to mass spectrometry enables a highly ef‐ ficient analysis even of crude samplesoffering all the possibilities of collision induced disso‐

0.0 2.0 4.0 6.0 8.0 10.0

Column, PA/PDA (Kap. 155, polymerised for 10 min), 80 x 0.2 mm; chromatographic conditions: mobile phase, A: 0.1 % TFA, B: ACN, 0.1 % TFA; gradient, 5-45 % B in 10 min; flow rate, 8 µl/min; temperature, RT; detection, UV 210 nm; Peak identification: (1) Hydroxytyrosol, (2) Tyrosol, (3) Caffeic acid, (4) Vanillin and

**Figure 5.** Separation of olive oil ingredients using a monolithic capillary column. Conditions: capillary 80 x 0.2 mm; mobile phase, A: 0.1% TFA; B: CAN; gradient, 5-45%B in 10 min; Flow rate 8 µl/min; temperature, RT; detection, UV

As an alternative separation method capillary electrophoresis (CE) and /or electrochroma‐ tography (CEC) can be applied. In CE separation of analytes is achieved due to their differ‐ ent ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm [4]. As has already been remarked the electroosmotic flow (EOF) has a main influence on the separation and can be used for speeding up. In CEC both an electrical field and high pressure are applied resulting in high resolution. This technique can be applied to check the identification and purity of standards compounds with very high efficiency. For the reproducible separation and analy‐ sis of food ingredients such as phenols, acids, peptides, lipids, coating of the capillary´s in‐ ner wall was shown being advantageous as irreversible analyte adsorption by free hydroxyl-groups from the silanole of the fused silica capillary can be avoided. Latex-diol

210 nm. Peak assignment, (1) hydroxytyrosol, (2) tyrosol, (3) caffeic acid, (4) vanillin, (5) oleuropein.

4 5

**time [min]**

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ciation (CID) and database search [15].

0

(5) Oleuropein.

2

4

6

8

10

12

1

2

3

**Figure 4.** Principle of material enhanced laser desorption ionization (MELDI)

For the analysis of low-molecular weight compounds (MW < 1000 Da) the conventional MELDI approach is replaced by the matrix-free (mf) MELDI approach for which the addi‐ tion of a matrix substance is not required so that no disturbing peaks appear. In this ap‐ proach a conventional steel target with a 50 nm thick titanium oxide layer can be applied fulfilling all requirements for a successful laser desorption ionization process [11].

As an alternative the incubated analytes of interest can be selectively eluted from the func‐ tinalised carrier material and further analysed by liquid chromatography (LC) or capillary electrophoresis (CE).

#### **2.3. Liquid chromatography, capillary electrophoresis and electrochromatography**

Novel materials used in miniaturised liquid chromatography (μ-LC) are mainly polymer based, e.g. poly(1,2-bis(p-vinylphenyl)ethane). These polymers possess the huge advantage that chemical (composition of the polymer) and physical parameters including mainly po‐ rosity can be adjusted [12]. Extensive investigations on polymerisation time and tempera‐ ture have been carried out enabling a tailored design of micro-, meso- and macro-pore distribution [13, 14]. This results in the applicability of such capillaries with an inner diame‐ ter between 20 and 200 μm for even the separation of high- and low-molecular weight com‐ pounds. These capillaries can be highly successfully applied analysing peptides, proteins, oligonucleotides, DNA fragments as well as "small molecules" such as phenols, flavonoids, catechins, acids etc. Figure 5 shows as an example the separation of olive oil ingredients. This separation is characterised by a very high ratio of flow to back pressure, which is of high interest to perform extremely rapid Coupling to mass spectrometry enables a highly ef‐ ficient analysis even of crude samplesoffering all the possibilities of collision induced disso‐ ciation (CID) and database search [15].

steel target used in MALDI-TOF/MS, a matrix substance is added (e.g., sinapinic acid) and finally the mass spectrum is generated by the laser desorption ionisation process. The result is a mass spectrum being characteristic for a patient and/or the nutrition profile. Multivari‐ ate analysis (MVA) can be applied for further data analysis and interpretation, a clustering into certain stages of an illness can be achieved, respectively. From the mass spectrum po‐ tent biomarker molecules can be selected and identified by further analytical steps. The bio‐ marker itself and/or the profile of the corresponding mass spectrum can be used for the screening of certain diseases, stages therefrom, allergies, nutrition effects and so on [7].

**Figure 4.** Principle of material enhanced laser desorption ionization (MELDI)

electrophoresis (CE).

182 Latest Research into Quality Control

For the analysis of low-molecular weight compounds (MW < 1000 Da) the conventional MELDI approach is replaced by the matrix-free (mf) MELDI approach for which the addi‐ tion of a matrix substance is not required so that no disturbing peaks appear. In this ap‐ proach a conventional steel target with a 50 nm thick titanium oxide layer can be applied

As an alternative the incubated analytes of interest can be selectively eluted from the func‐ tinalised carrier material and further analysed by liquid chromatography (LC) or capillary

Novel materials used in miniaturised liquid chromatography (μ-LC) are mainly polymer based, e.g. poly(1,2-bis(p-vinylphenyl)ethane). These polymers possess the huge advantage that chemical (composition of the polymer) and physical parameters including mainly po‐

fulfilling all requirements for a successful laser desorption ionization process [11].

**2.3. Liquid chromatography, capillary electrophoresis and electrochromatography**

**Figure 5.** Separation of olive oil ingredients using a monolithic capillary column. Conditions: capillary 80 x 0.2 mm; mobile phase, A: 0.1% TFA; B: CAN; gradient, 5-45%B in 10 min; Flow rate 8 µl/min; temperature, RT; detection, UV 210 nm. Peak assignment, (1) hydroxytyrosol, (2) tyrosol, (3) caffeic acid, (4) vanillin, (5) oleuropein.

(5) Oleuropein.

As an alternative separation method capillary electrophoresis (CE) and /or electrochroma‐ tography (CEC) can be applied. In CE separation of analytes is achieved due to their differ‐ ent ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm [4]. As has already been remarked the electroosmotic flow (EOF) has a main influence on the separation and can be used for speeding up. In CEC both an electrical field and high pressure are applied resulting in high resolution. This technique can be applied to check the identification and purity of standards compounds with very high efficiency. For the reproducible separation and analy‐ sis of food ingredients such as phenols, acids, peptides, lipids, coating of the capillary´s in‐ ner wall was shown being advantageous as irreversible analyte adsorption by free hydroxyl-groups from the silanole of the fused silica capillary can be avoided. Latex-diol and fullerene coated capillaries were successfully introduced and as a detection system online hyphenation to MALDI-TOF/MS was shown to be highly efficient not only for the in‐ vestigation of flavonoids but also for peptides, especially phosphorylated (Figure 6) [16, 17]. This system can be used for the investigation of the casein profile in milk offering the ad‐ vantage over all other more classical analysis tools that in this case also higher phosphory‐ lated species can be separated and detected. From the ratio of different phosphorylation degrees several interpretations concerning the quality but also the origin of the milk can be carried out.

analyse data. These are mainly multivariate (MVA) methods allowing to correct baseline, at‐ mospheric noise etc. For qualitative analysis in most cased principal component analysis

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An impressive example for the successful implementation in the food related production is the quality control of wine. It has been shown that NIR can be used to identify grapes, vines, age by qualitative (Figure 8) and its ingredients (acids, carbohydrates, pH etc.) simultane‐ ously, non-invasively within a few seconds by quantitative analysis [19]. Another big ad‐ vantage of this method can be found by the fact that the sample is not destroyed and can

Quantitative NIRS methods, which allow determining the carbohydrate, total acid, tartaric acid, malic acid, pH in grape variety and the polyphenol content in grapes were established [19]. The method can control the quality already at a very early stage during the wine pro‐ duction and allows improvement of its quality by this. Grapes of 12 different vines (*Weißburgunder, Chardonnay, Ruländer, Silvaner, Müller Thurgau, Gewürztraminer, Sauvignon, Lagrein, Grossvernatsch, Blauburgunder, Cabernet, Merlot*) were harvested in autumn 2000 and squeezed. The obtained grape variety was thermo stated at 23°C and analyzed quantitative‐ ly by NIRS in the transflection mode using an optical thin layer thickness of 1 mm. In order to establish a calibration model 252 spectra of samples with lower and upper concentration as a reference were recorded. 76 % of all spectra were randomly used for calibration, 24% for validation. Data preparation was carried out in order to minimize technical influences,

therefore be used for further purposes including following analytical steps.

(PCA), for quantitative partial least square regression (PLSR) are applied [18].

**Figure 7.** Sample measurement modes in NIR

**Figure 6.** Inner capillary wall coatings applied in CE and CEC

#### **2.4. Vibrational spectroscopy**

For quality control both mid- (MIR, 400 – 4000 cm-1) and near-infrared (NIR, 4000 – 12000 cm-1) can be conducted. In MIR fundamental stretching and bending vibrations occur, in NIR the corresponding overtones and combination vibrations are detected. This means that NIR-spectra can contain a lot of more vibrational information, which is an advantage for the analysis of highly complex samples. Therefore, during the last decade several applications in the field of food analysis were developed in the NIR region. Samples can be analysed ei‐ ther in transmission, reflectance and interactance mode (Figure 7) so that liquid as well as solid samples can be investigated. Due to the quite broad bands compared to MIR, chemo‐ metrical spectra treatment is required for establishing adequate calibration models and to analyse data. These are mainly multivariate (MVA) methods allowing to correct baseline, at‐ mospheric noise etc. For qualitative analysis in most cased principal component analysis (PCA), for quantitative partial least square regression (PLSR) are applied [18].

**Figure 7.** Sample measurement modes in NIR

and fullerene coated capillaries were successfully introduced and as a detection system online hyphenation to MALDI-TOF/MS was shown to be highly efficient not only for the in‐ vestigation of flavonoids but also for peptides, especially phosphorylated (Figure 6) [16, 17]. This system can be used for the investigation of the casein profile in milk offering the ad‐ vantage over all other more classical analysis tools that in this case also higher phosphory‐ lated species can be separated and detected. From the ratio of different phosphorylation degrees several interpretations concerning the quality but also the origin of the milk can be

For quality control both mid- (MIR, 400 – 4000 cm-1) and near-infrared (NIR, 4000 – 12000 cm-1) can be conducted. In MIR fundamental stretching and bending vibrations occur, in NIR the corresponding overtones and combination vibrations are detected. This means that NIR-spectra can contain a lot of more vibrational information, which is an advantage for the analysis of highly complex samples. Therefore, during the last decade several applications in the field of food analysis were developed in the NIR region. Samples can be analysed ei‐ ther in transmission, reflectance and interactance mode (Figure 7) so that liquid as well as solid samples can be investigated. Due to the quite broad bands compared to MIR, chemo‐ metrical spectra treatment is required for establishing adequate calibration models and to

carried out.

184 Latest Research into Quality Control

**Figure 6.** Inner capillary wall coatings applied in CE and CEC

**2.4. Vibrational spectroscopy**

An impressive example for the successful implementation in the food related production is the quality control of wine. It has been shown that NIR can be used to identify grapes, vines, age by qualitative (Figure 8) and its ingredients (acids, carbohydrates, pH etc.) simultane‐ ously, non-invasively within a few seconds by quantitative analysis [19]. Another big ad‐ vantage of this method can be found by the fact that the sample is not destroyed and can therefore be used for further purposes including following analytical steps.

Quantitative NIRS methods, which allow determining the carbohydrate, total acid, tartaric acid, malic acid, pH in grape variety and the polyphenol content in grapes were established [19]. The method can control the quality already at a very early stage during the wine pro‐ duction and allows improvement of its quality by this. Grapes of 12 different vines (*Weißburgunder, Chardonnay, Ruländer, Silvaner, Müller Thurgau, Gewürztraminer, Sauvignon, Lagrein, Grossvernatsch, Blauburgunder, Cabernet, Merlot*) were harvested in autumn 2000 and squeezed. The obtained grape variety was thermo stated at 23°C and analyzed quantitative‐ ly by NIRS in the transflection mode using an optical thin layer thickness of 1 mm. In order to establish a calibration model 252 spectra of samples with lower and upper concentration as a reference were recorded. 76 % of all spectra were randomly used for calibration, 24% for validation. Data preparation was carried out in order to minimize technical influences, which mainly cause a drift in baseline. Quantitative analysis was carried out by partial least square regression (PLSR).

ric acid content in grape variety with an absolute error of estimation of 0.40 g/l and predic‐

*Malic acid.* Malic acid often shows 2-5 times higher values compared to tartaric acid. Calibra‐ tion between 2.9 and 7.0 g/l after normalization between 0 and 1 and calculation of a second smoothened derivative was carried out using three factors, SEE and SEP showing acceptable agreement.Absolute values for SEE, and BIAS were 0.43 g/l and -4.25×10-15. Straight line for

*pH.* Normalization and calculation of the smoothed 2nd derivative between 4500 and 7308 cm-1 showed an optimum for BIAS at five factors. Despite the narrow calibration range of

In order to enable the determination of these parameters with only one single measurement, simultaneous analysis of the carbohydrate, total acid, tartaric acid, malic acid content and pH was achieved by performing normalization (between 0 and 1) and calculating its 2nd de‐ rivative (Taylor 3 points). Four factors over a wavenumber range from 4500 to 7308 cm-1 showed 73-100% agreement between SEE and SEP. Linear regression showed high linearity

described single analysis this method allows a quantitative analysis of all parameters at once

Polyphenols mainly influence taste, sensory properties and color of a wine. Therefore, a rap‐ id method to analyze its quantity is important. The method according to Folin - Ciocalteu was used as a reference method (see Materials and Methods). Gallic acid-1-hydrate was used as reference standard in a concentration range from 0 to 4.93 μg/ml with equidistant steps. 24 gallic acid-1-hydrate solutions in a concentration range between 0.442 and 7.08 mg/ml were measured in the transmission mode threefold and in random order by NIRS. Evaluation using PLSR was achieved by dividing 72-recorded spectra randomly into a cali‐ bration (54 spectra) and validation (18 spectra) set. Data pretreatment comprised normaliza‐ tion between 0 and 1 and calculation of the 1st derivative (Savitzky-Golay) between 4008-7512 cm-1. Using three factors, the PRESS function showed a minimum and a good agreement between SEE (0.45 mg/ml) and SEP (0.46 mg/ml). Linear regression between pre‐ dicted and true values allowed to predict the gallic acid-1-hydrate concentration between 0

In order to determine the total polyphenol concentration 30 must samples were measured in the transmission mode threefold and in random order. 90 spectra were divided into 72 cali‐ bration and 18 validation spectra. Normalization and performing of the 1st derivative al‐ lowed minimizing shifts in the baseline. 4 factors were necessary to obtain a minimum for the PRESS function and to get a maximum agreement of SEE and SEP. Linear regression al‐ lowed correlating true and predicted values with a R2 of 0.97. Compared to the traditionally used Folin - Ciocalteu method in a winery, which is very time-consuming and expensive due to the usage of different chemicals, the NIRS method is very simple, precise and incom‐

of 0.82.

=0.89 and allowed a prediction of the malic acid content

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. Compared to the above-

tion of 0.54 g/l.

calibration showed a linearity of R2

pH 3.09 - 3.74 the calibration equation shows a R2

for each investigated parameter with slightly lower values for R2

within a few seconds. Values for SEP are slightly increased (Table 1).

with an absolute error of 0.55 g/l.

and 7 mg/ml with R2

parably fast.

=0.98.

**Figure 8.** Factor plot of 141 specta of different wines (Lagrein, Chianti, Cabernet Sauvignon). Conditions: Normalisa‐ tion, 1. derivative; wavenumber range, 4500 - 10000 cm-1; thickness 3 mm; scans, 10; temperature, 23°C.

*Carbohydrates.* Data preparation comprised normalization between 0 and 1 and following calculation of the first derivative using a wavenumber range from 4500 - 7548 cm-1. The PRESS function showed that 3 factors were needed for the calculation of the model. Calcula‐ tion with 3 factors resulted in a good conformity between SEE and SEP. Linear regression between true and predicted values resulted in a value for the correlation coefficient of R2 =0.99 for calibration and R2 =0.99 for validation. Results for SEE and SEP: 0.13° KMW and 0.11 °KMW, the BIAS value is 2.30×10-15.

*Total acids.* PLSR in a concentration range between 5 and 11 g/l included normalization be‐ tween 0 and 1, full multiplicative scatter correction (MSC) and calculation of the 1st deriva‐ tive (Taylor 3 points) between 4500 and 7548 cm-1. 3 factors were necessary to obtain a minimum for PRESS and an agreement between SEE (0.60 g/l) and SEP (0.61 g/l) of nearly 100%.The highly linear model allows determining the total acid content with a prediction er‐ ror of 0.61 g/l.

*Tartaric acid.* After normalization, performing of the 1st derivative over between 4500 and 7308 cm-1, four factors were used for creation of the highly linear model depicted in Figure 6c with R2 =0.91 for calibration and R2 =0.87 for validation). Despite the small concentration range between 3.1 and 6.7 g/l used for calibration this system allows to determine the tarta‐ ric acid content in grape variety with an absolute error of estimation of 0.40 g/l and predic‐ tion of 0.54 g/l.

which mainly cause a drift in baseline. Quantitative analysis was carried out by partial least

**Figure 8.** Factor plot of 141 specta of different wines (Lagrein, Chianti, Cabernet Sauvignon). Conditions: Normalisa‐

*Carbohydrates.* Data preparation comprised normalization between 0 and 1 and following calculation of the first derivative using a wavenumber range from 4500 - 7548 cm-1. The PRESS function showed that 3 factors were needed for the calculation of the model. Calcula‐ tion with 3 factors resulted in a good conformity between SEE and SEP. Linear regression between true and predicted values resulted in a value for the correlation coefficient of

*Total acids.* PLSR in a concentration range between 5 and 11 g/l included normalization be‐ tween 0 and 1, full multiplicative scatter correction (MSC) and calculation of the 1st deriva‐ tive (Taylor 3 points) between 4500 and 7548 cm-1. 3 factors were necessary to obtain a minimum for PRESS and an agreement between SEE (0.60 g/l) and SEP (0.61 g/l) of nearly 100%.The highly linear model allows determining the total acid content with a prediction er‐

*Tartaric acid.* After normalization, performing of the 1st derivative over between 4500 and 7308 cm-1, four factors were used for creation of the highly linear model depicted in Figure

range between 3.1 and 6.7 g/l used for calibration this system allows to determine the tarta‐

tion, 1. derivative; wavenumber range, 4500 - 10000 cm-1; thickness 3 mm; scans, 10; temperature, 23°C.

Cabernet Sauvignon

=0.99 for validation. Results for SEE and SEP: 0.13° KMW and

=0.87 for validation). Despite the small concentration

Chianti

square regression (PLSR).

186 Latest Research into Quality Control

Lagrein

=0.99 for calibration and R2

0.11 °KMW, the BIAS value is 2.30×10-15.

=0.91 for calibration and R2

R2

ror of 0.61 g/l.

6c with R2

*Malic acid.* Malic acid often shows 2-5 times higher values compared to tartaric acid. Calibra‐ tion between 2.9 and 7.0 g/l after normalization between 0 and 1 and calculation of a second smoothened derivative was carried out using three factors, SEE and SEP showing acceptable agreement.Absolute values for SEE, and BIAS were 0.43 g/l and -4.25×10-15. Straight line for calibration showed a linearity of R2 =0.89 and allowed a prediction of the malic acid content with an absolute error of 0.55 g/l.

*pH.* Normalization and calculation of the smoothed 2nd derivative between 4500 and 7308 cm-1 showed an optimum for BIAS at five factors. Despite the narrow calibration range of pH 3.09 - 3.74 the calibration equation shows a R2 of 0.82.

In order to enable the determination of these parameters with only one single measurement, simultaneous analysis of the carbohydrate, total acid, tartaric acid, malic acid content and pH was achieved by performing normalization (between 0 and 1) and calculating its 2nd de‐ rivative (Taylor 3 points). Four factors over a wavenumber range from 4500 to 7308 cm-1 showed 73-100% agreement between SEE and SEP. Linear regression showed high linearity for each investigated parameter with slightly lower values for R2 . Compared to the abovedescribed single analysis this method allows a quantitative analysis of all parameters at once within a few seconds. Values for SEP are slightly increased (Table 1).

Polyphenols mainly influence taste, sensory properties and color of a wine. Therefore, a rap‐ id method to analyze its quantity is important. The method according to Folin - Ciocalteu was used as a reference method (see Materials and Methods). Gallic acid-1-hydrate was used as reference standard in a concentration range from 0 to 4.93 μg/ml with equidistant steps. 24 gallic acid-1-hydrate solutions in a concentration range between 0.442 and 7.08 mg/ml were measured in the transmission mode threefold and in random order by NIRS. Evaluation using PLSR was achieved by dividing 72-recorded spectra randomly into a cali‐ bration (54 spectra) and validation (18 spectra) set. Data pretreatment comprised normaliza‐ tion between 0 and 1 and calculation of the 1st derivative (Savitzky-Golay) between 4008-7512 cm-1. Using three factors, the PRESS function showed a minimum and a good agreement between SEE (0.45 mg/ml) and SEP (0.46 mg/ml). Linear regression between pre‐ dicted and true values allowed to predict the gallic acid-1-hydrate concentration between 0 and 7 mg/ml with R2 =0.98.

In order to determine the total polyphenol concentration 30 must samples were measured in the transmission mode threefold and in random order. 90 spectra were divided into 72 cali‐ bration and 18 validation spectra. Normalization and performing of the 1st derivative al‐ lowed minimizing shifts in the baseline. 4 factors were necessary to obtain a minimum for the PRESS function and to get a maximum agreement of SEE and SEP. Linear regression al‐ lowed correlating true and predicted values with a R2 of 0.97. Compared to the traditionally used Folin - Ciocalteu method in a winery, which is very time-consuming and expensive due to the usage of different chemicals, the NIRS method is very simple, precise and incom‐ parably fast.

biological tissues and the modern analytical techniques enabling molecular imaging of com‐ plex samples. These techniques are based on the absorption of IR radiations by vibrational transitions in covalent bonds and their major advantage is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue by avoiding time-consuming extraction, purification and separation steps. These new techniques enable global analysis of biological samples with high spatial resolution and provide unique chemi‐ cal-morphological information about the tissue status. With these non-destructive examina‐ tion methods it is possible to get qualitative and quantitative information of heterogeneous

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Additionally, MALDI-TOF/MS imaging can be applied from the same sample of interest to get knowledge concerning the molecular weight distribution. This method is also suitable for studying the effect of nutrition onto different kinds of diseases, e.g. prostate cancer.

The techniques described can be applied according to the scheme depicted in Figure 1. This systematic analytical strategy allows getting multifacial knowledge and insights into food

samples.

**Figure 9.** Principle of hyperspectral cube

and samples derived therefrom.

**3. Conclusions**

**Table 1.** Prediction results for the determination of the carbohydrate, total acid, tartaric acid, malic acid content and pH

Quality control of coffee ingredients including caffeine, theobromine and theophylline [20] and of food additives deriving from the highly interesting field of Traditional Chinese Medi‐ cine (TCM) [21] can be carried out in a similar way. Thereby, emphasis must be put onto the calibration method for which the above mentioned techniques can be applied as a reference. A new analytical method based on near infrared spectroscopy (NIRS) for the quantitation of the three main alkaloids caffeine (Caf), theobromine (Tbr) and theophylline (Tph) in roasted coffee after discrimination of the rough green beans into Arabic and Robusta was establish‐ ed. This validated method was compared to the most commonly used liquid chromatogra‐ phy (LC) connected to UV and mass spectrometric (MS) detection. As analysis time plays an important role in choosing a reference method for the calibration of the NIR-spectrometer, the non-porous silica-C18 phase offers a very fast method. Coupling of the optimised LC method to a mass spectrometer (MS) via an electrospray ionisation (ESI) interface not only allowed to identify Caf, Tbr and Tph by their characteristic fragmentation pattern using col‐ lisionally induced dissociation (CID), but also to quantitate the content of the three analytes, which was found to be 6% higher compared to UV-detection. The validated LC–UV method was chosen as a reference method for the calibration of the NIRS system. Analysis of 83 liq‐ uid coffee extracts in random order resulted for Caf and Tbr in values for S.E.E. (standard error of estimation) of 0.34, 0.40 g/100 g, S.E.P. (standard error of prediction) of 0.07 and 0.10 g/100 g with correlation coefficients of 0.86 and 0.85 in a concentration range between 0.10 and 4.13 g/100 g. Compared to LC the lower limit of detection (LOD) of the NIRS-method is found at 0.05 g/100 g compared to 0.244–0.60 ng/100 g in LC, which makes it impossible to analyse Tph by NIRS.

The possibility to hyphenate a MIR/NIR spectrometer to a microscope unit allows determining the distribution of active ingredients within a tissue sample down to a resolution of 1.2 μm [22]. A "hyperspectral cube" is recorded with the dimensions of the sample on the x- and y-axis and the absorbance on the z-axis from which the image can be extracted (Figure 9).

Fourier Transform Infrared (FTIR) spectroscopic imaging and mapping techniques have be‐ come essential tools for the detection and characterization of the molecular components of biological tissues and the modern analytical techniques enabling molecular imaging of com‐ plex samples. These techniques are based on the absorption of IR radiations by vibrational transitions in covalent bonds and their major advantage is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue by avoiding time-consuming extraction, purification and separation steps. These new techniques enable global analysis of biological samples with high spatial resolution and provide unique chemi‐ cal-morphological information about the tissue status. With these non-destructive examina‐ tion methods it is possible to get qualitative and quantitative information of heterogeneous samples.

**Figure 9.** Principle of hyperspectral cube

Additionally, MALDI-TOF/MS imaging can be applied from the same sample of interest to get knowledge concerning the molecular weight distribution. This method is also suitable for studying the effect of nutrition onto different kinds of diseases, e.g. prostate cancer.

#### **3. Conclusions**

**Parameter Unit SEE SEP BIAS**

Note. a Single analysis; b Simultaneous analysis

188 Latest Research into Quality Control

pH

analyse Tph by NIRS.

 Prediction results for the determination of the carbohydrate, total acid, tartaric acid, malic acid content and

Quality control of coffee ingredients including caffeine, theobromine and theophylline [20] and of food additives deriving from the highly interesting field of Traditional Chinese Medi‐ cine (TCM) [21] can be carried out in a similar way. Thereby, emphasis must be put onto the calibration method for which the above mentioned techniques can be applied as a reference. A new analytical method based on near infrared spectroscopy (NIRS) for the quantitation of the three main alkaloids caffeine (Caf), theobromine (Tbr) and theophylline (Tph) in roasted coffee after discrimination of the rough green beans into Arabic and Robusta was establish‐ ed. This validated method was compared to the most commonly used liquid chromatogra‐ phy (LC) connected to UV and mass spectrometric (MS) detection. As analysis time plays an important role in choosing a reference method for the calibration of the NIR-spectrometer, the non-porous silica-C18 phase offers a very fast method. Coupling of the optimised LC method to a mass spectrometer (MS) via an electrospray ionisation (ESI) interface not only allowed to identify Caf, Tbr and Tph by their characteristic fragmentation pattern using col‐ lisionally induced dissociation (CID), but also to quantitate the content of the three analytes, which was found to be 6% higher compared to UV-detection. The validated LC–UV method was chosen as a reference method for the calibration of the NIRS system. Analysis of 83 liq‐ uid coffee extracts in random order resulted for Caf and Tbr in values for S.E.E. (standard error of estimation) of 0.34, 0.40 g/100 g, S.E.P. (standard error of prediction) of 0.07 and 0.10 g/100 g with correlation coefficients of 0.86 and 0.85 in a concentration range between 0.10 and 4.13 g/100 g. Compared to LC the lower limit of detection (LOD) of the NIRS-method is found at 0.05 g/100 g compared to 0.244–0.60 ng/100 g in LC, which makes it impossible to

The possibility to hyphenate a MIR/NIR spectrometer to a microscope unit allows determining the distribution of active ingredients within a tissue sample down to a resolution of 1.2 μm [22]. A "hyperspectral cube" is recorded with the dimensions of the sample on the x- and y-axis and

Fourier Transform Infrared (FTIR) spectroscopic imaging and mapping techniques have be‐ come essential tools for the detection and characterization of the molecular components of

the absorbance on the z-axis from which the image can be extracted (Figure 9).

**a b a b a b**

The techniques described can be applied according to the scheme depicted in Figure 1. This systematic analytical strategy allows getting multifacial knowledge and insights into food and samples derived therefrom.

#### **Acknowledgements**

This work was financially supported by the Efre-project "Originalp" (EU) and Agrarmarket‐ ing Austria (AMA, Vienna, Austria).

[9] Rainer, M., Sonderegger, H., Bakry, R., Huck, C. W., Morandell, S., Huber, L. A., Gjerde, D. T., & Bonn, G. K. Analysis of protein phosphorylation by monolithic ex‐ traction columns based on poly(divinylbenzene) containing embedded titanium di‐ oxide and zirconium dioxide nano-powders. Proteomics (2008). , 8(21), 4593-4602. [10] Hahn, H., Rainer, M., Ringer, T., Gjerde, D., Huck, C. W., & Bonn, G. K. Ultra-fast Microwave-Assisted In-Tip Digestion of Proteins. J. Proteom. Res. (2009). , 8(9),

Novel Analytical Tools for Quality Control in Food Science

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191

[11] Bonn, G. K., Bakry, R., Huck, C. W., Vallant, R., & Szabo, Z. Analysis of low molecu‐ lar weight molecules by maldi-ms. Eur. Pat. Appl. ((2008). pp. CODEN: EPXXDW EP

[12] Greiderer A., Clark Ligon S.Jr., Huck C.W., Bonn G.K.Monolithic poly( -bis(p-vinyl‐ phenyl)ethane capillary columns for simultaneous separation of low- and high-mo‐

[13] Greiderer A., Trojer L., Huck C.W., Bonn G.K.Influence of the Polymerisation time on the Porous and Chromatographic Properties of Monolithic Poly(1,2-bis(p-vinyl‐ phenyl)ethane Capillary Columns.J.Chromatogr. A (2009). , 1216(45), 7747-7754. [14] Heigl, N., Greiderer, A., Petter, C. H., Siesler, H. W., Bonn, G. K., & Huck, C. W. Si‐ multaneous Determination of Physical and Chemical Parameters of Monolithic Po‐ rous Polymers with a Combined Use of Fourier-Transform Near Infrared Diffuse Reflection Spectroscopy and Multivariate Techniques. Anal. Chem. (2008). , 80(22),

[15] Trojer, L., Greiderer, A., Bisjak, C. P., Wieder, W., Heigl, N., Huck, C. W., Bonn, G. K., in, Handbook., of, H. P. L. C., Second, Edition., edited, by., Corradini, D., &

[16] Bachmann S., Vallant R., Bakry R., Huck C.W., Corradini D., Bonn G.K.Capillary electrophoresis coupled to matrix assisted laser desorption ionization with novel co‐

[17] Stöggl W.M., Huck C.W., Stecher G., Bonn G.K.Capillary Electrochromatography of

[18] Petter C.H., Heigl N., Bachmann S., Huck-Pezzei V.A.C., Najam-ul-Haq M., Bakry R., Bernkop-Schnürch A., Bonn G.K., Huck C.W.Near Infrared Spectroscopy Compared to Liquid Chromatography Coupled to Mass Spectrometry and Capillary Electropho‐ resis as a Detection Tool for Peptide Reaction Monitoring.Amino Acids, (2008). ,

[19] Guggenbichler, W., Huck, C. W., Kobler, A., Bonn, G. K., Near, Infrared., Reflectance, Spectroscopy., Tool, A., for, Quality., Control, In., & Wine, Production. J. Food Agric.

[20] Huck C.W., Guggenbichler W., Bonn G.K., Analysis of Caffeine, Theobromine and Theophylline in Coffee by Near Infrared Reflectance Spectroscopy (NIRS) Compared

Press, . (2010). Print 978-1-57444-554-1eBook ISBN: 978-1-4200-1694-9, 3-45.

valently coated capillaries.Electrophoresis, (2010). , 31, 618-629.

Biologically Relevant Flavonoids.Electrophoresis, (2006). , 27-787.

4225-4230.

8493-8500.

34(4), 605-616.

Environm., (2006). , 4(2), 98-106.

1973142 A1 20080924 AN 2008:1151584

lecular weight compounds.J.Sep. Sci. (2009).

#### **Author details**

Christian W. Huck

Institute of Analytical Chemistry and Radiochemistry, CCB – Center for Chemistry and Bio‐ medicine, Innsbruck, Austria

#### **References**

[9] Rainer, M., Sonderegger, H., Bakry, R., Huck, C. W., Morandell, S., Huber, L. A., Gjerde, D. T., & Bonn, G. K. Analysis of protein phosphorylation by monolithic ex‐ traction columns based on poly(divinylbenzene) containing embedded titanium di‐ oxide and zirconium dioxide nano-powders. Proteomics (2008). , 8(21), 4593-4602.

**Acknowledgements**

190 Latest Research into Quality Control

**Author details**

Christian W. Huck

**References**

medicine, Innsbruck, Austria

ing Austria (AMA, Vienna, Austria).

This work was financially supported by the Efre-project "Originalp" (EU) and Agrarmarket‐

Institute of Analytical Chemistry and Radiochemistry, CCB – Center for Chemistry and Bio‐

[1] Huck C.W., Popp M., Scherz H., Bonn G.K.Development and Evaluation of a New Method for the Determination of the Carotenoid Content in Selected Vegetables by

[2] Stöggl, W. M., Huck, C. W., Scherz, H., Bonn, G. K., Analysis, of., Vitamin, E., in, Food., Phytopharmaceutical, Preparations., by, H. P. L. C., -A, H. P. L. C., -M, P. C. I.,

[3] JakschitzT., HuckC.W., LubbadS., Bonn G.K.Monolithic Poly(TMSM-DMSBMS) Sta‐ tionary Phases for the fast Separation of Oligonucleotides and Proteins.J. Chroma‐

[4] Huck C.W., Bakry R., Bonn G.K.Progress in capillary electrophoresis of biomarkers

[5] Stöggl W.M., Huck C.W., Stecher G., Bonn G.K., Capillary Electrochromatography of

[6] Huck C.W., Bonn G.K.Review: Polymer based sorbents for solid-phase extraction.J.

[7] Feuerstein I., Najam-ul-Haq M., Rainer M., Trojer L., Bakry R., Hidayat Aprilita N., Stecher G., Huck C.W., Klocker H., Bartsch G., Guttman A., BonnG.K.Material En‐ hanced Laser Desorption/Ionization (MELDI)- a new protein profiling tool utilizing specific carrier materials for TOF-MS Analysis.J. Am. Soc. Mass Spectrom. (2006). ,

[8] Petter C.H., Heigl N., Bachmann S., Huck-Pezzei V.A.C., Najam-ul-Haq M., Bakry R., Bernkop-Schnürch A., Bonn G.K., Huck\*C.W.Near Infrared Spectroscopy Compared to Liquid Chromatography Coupled to Mass Spectrometry and Capillary Electropho‐

resis as a Detection Tool for Peptide Reaction Monitoring.Amino Acids (2008).

and metabolites between 2002 and 2005.Electrophoresis (2006). , 27-111.

Biologically Relevant Flavonoids. (2006). *Electrophoresis*, 27-787.

HPLC and HPLC-MS/MS.J. Chromatogr. Sci. (2000). , 38-441.

& , S. M. S. Chromatographia (2001). , 54-179.

togr. A (2007). , 1147-53.

Chromatogr. A (2000). , 885-51.

17-1203.

to High Performance Liquid Chromatography (HPLC) Coupled to Mass Spectrome‐ try.Anal. Chim. Acta, (2005). , 538-195.

**Section 3**

**Quality Control in Pharmaceutics**

**Quality Control in Pharmaceutics**

to High Performance Liquid Chromatography (HPLC) Coupled to Mass Spectrome‐

[21] Mattle, C., Heigl, N., Bonn, G. K., & Huck, C. W. Near Infrared Diffuse Reflection Spectroscopy and Multivariate Calibration Hyphenated to Thin Layer Chromatogra‐ phy for Quality Control and Simultaneous Quantification of Methoxylated Flavones

[22] Pezzei, C., Pallua, J. D., Schaefer, G., Seifarth, C., Huck-Pezzei, V., Bittner, L. K., Klocker, H., Bartsch, G., Bonn, G. K., Huck, C. W., Prostate, cancer., characterization, by., Fourier, Transform., & infrared, microspectroscopy. Mol. Biosys., (2010). , 6-2287.

in a Phytomedicine. J. Planar Chromatogr., (2010). , 23(5), 348-352.

try.Anal. Chim. Acta, (2005). , 538-195.

192 Latest Research into Quality Control

**Chapter 9**

**Microbial Quality**

http://dx.doi.org/10.5772/52114

**1. Introduction**

and biological product.

pharmaceutical product.

packaging in its final container and labeling." [3]

properly cited.

**Concerns for Biopharmaceuticals**

Farzaneh Lotfipour and Somayeh Hallaj-Nezhadi

Finding an appropriate definition or a clear classification for biologically occurring pharma‐ ceutical products is a complicated task because of overlapping borders and consequent mis‐ conceptions in this area. Indeed, numerous definitions and classifications for this category of products have been proposed so far, and different points of view for this concept can be

To obtain a better view of biopharmaceutical concept, first, it is necessary to know the present definitions for the main constituents of the word, that is, pharmaceutical product

According to the WHO, a finished pharmaceutical product (FPP) is "A finished dosage form of a pharmaceutical product, which has undergone all stages of manufacture, including

An active pharmaceutical ingredient can be defined as "A substance used in a finished phar‐ maceutical product (FPP), intended to furnish pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings." [3] Hence, in brief, it can be said that any material, regardless of its origin or structure, with treatment, diagnosis, or prevention applications and passing regulatory requirements, is a

On the other hand, the FDA definition for biological products is as follows: "Biological products or biologics are medical products made from a variety of natural sources (human, animal or microorganism). Like drugs, some biologics are intended to treat diseases and

> © 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

found in research literature, business, industry, and even the general public [1, 2].

Additional information is available at the end of the chapter

### **Chapter 9**

## **Microbial Quality Concerns for Biopharmaceuticals**

Farzaneh Lotfipour and Somayeh Hallaj-Nezhadi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52114

#### **1. Introduction**

Finding an appropriate definition or a clear classification for biologically occurring pharma‐ ceutical products is a complicated task because of overlapping borders and consequent mis‐ conceptions in this area. Indeed, numerous definitions and classifications for this category of products have been proposed so far, and different points of view for this concept can be found in research literature, business, industry, and even the general public [1, 2].

To obtain a better view of biopharmaceutical concept, first, it is necessary to know the present definitions for the main constituents of the word, that is, pharmaceutical product and biological product.

According to the WHO, a finished pharmaceutical product (FPP) is "A finished dosage form of a pharmaceutical product, which has undergone all stages of manufacture, including packaging in its final container and labeling." [3]

An active pharmaceutical ingredient can be defined as "A substance used in a finished phar‐ maceutical product (FPP), intended to furnish pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings." [3] Hence, in brief, it can be said that any material, regardless of its origin or structure, with treatment, diagnosis, or prevention applications and passing regulatory requirements, is a pharmaceutical product.

On the other hand, the FDA definition for biological products is as follows: "Biological products or biologics are medical products made from a variety of natural sources (human, animal or microorganism). Like drugs, some biologics are intended to treat diseases and

properly cited.

© 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

medical conditions or to prevent or diagnose diseases." [4] Consequently, any product of bi‐ ological origin with treatment, diagnosis, or prevention applications is a biological product.

A biotechnology-derived product is another concept that should be taken into consideration. It is defined by Walsh as "any pharmaceutical product used for a therapeutic or in vivo di‐ agnostic purpose, which is produced in full or in part by either traditional or modern bio‐ technological means." [5]

On comparing the definitions for a biotechnology-derived product and a biological product, both of which should be of biological origin, it is obvious that the key element in the former definition is the application of biotechnological means for production.

In practice, the regulatory requirements needed for a biotechnology-derived product and a biological product are methodologically different from the pharmaceutical product due to their biological essence. For example, the determination of adventitious agents such as vi‐ ruses, transmitting spongiform encephalopathy (TSE), and mycoplasma are included in most of the related guidelines and pharmacopeias for a biotechnology-derived product and a biological product.

**Figure 1.** The schematic representation for biotechnology-derived products and biopharmaceutical categories and

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197

The main category of biopharmaceuticals is manufactured via recombinant DNA technolo‐ gy. Indeed, recombinant DNA technologies are enabling techniques that manipulate and en‐ gineer different gene fragments and which have been introduced less than 50 years ago by

DNA and RNA extraction from different cell types, cutting DNA fragments using restriction endonucleases, joining DNA fragments by DNA ligases, PCR to amplify gene fragments, cloning of the gene fragments into different vectors, introduction of recombinant constructs into proper hosts, protein expression, extraction, and purification are some of the most

Figure 2 schematically represents the summarized process of production of a recombinant protein. As can be seen from the chart, first, the gene of interest should be isolated and am‐ plified from the original cell. According to the type of the cell, it can be done through direct total DNA extraction followed by a PCR using proper primers to obtain the gene in prokar‐ yotes. On the other hand, in eukaryotes, due to the existence of introns and some modifica‐ tions that occur in the transcribed mRNA, the process is considerably complicated. Introns are non-coding sequences which are removed after transcription versus coding sequences

the revolutionary invention of Polymerase Chain Reaction (PCR) by Kary Mullis [7].

the overlapping areas between them.

*2.1.1. Recombinant DNA technology*

**2. Methods for biopharmaceutical production**

**2.1. Production of an original recombinant system**

widely used means in recombinant protein production.

Finally, a biopharmaceutical is defined by Walsh as "A protein or nucleic acid based phar‐ maceutical substance used for therapeutic or in vivo diagnostic purposes, which is produced by means other than direct extraction from a native (non-engineered) biological source." This definition that will be used in the present chapter for biopharmaceuticals includes all pharmaceutical products produced by modern biotechnology techniques as well as nucleic acid (DNA or RNA) based pharmaceutical products for gene therapy. Hence, the overlap‐ ping area between biotechnology-derived products and biopharmaceuticals is the applica‐ tion of modern biotechnological means in their production. However, the differentiating area can be the application of traditional biotechnological means for the production of bio‐ technology-derived products. In addition, nucleic acid-based pharmaceutical products that are categorized as biopharmaceuticals are not biotechnology-derived products [6]. Figure 1 illustrates these overlapping and differentiating areas. Moreover, some examples of prod‐ ucts in these categories are shown in Table1.

**Table 1.** Some examples of products related to biological, biotechnology-derived, and biopharmaceutical products.

**Figure 1.** The schematic representation for biotechnology-derived products and biopharmaceutical categories and the overlapping areas between them.

### **2. Methods for biopharmaceutical production**

#### **2.1. Production of an original recombinant system**

#### *2.1.1. Recombinant DNA technology*

medical conditions or to prevent or diagnose diseases." [4] Consequently, any product of bi‐ ological origin with treatment, diagnosis, or prevention applications is a biological product.

A biotechnology-derived product is another concept that should be taken into consideration. It is defined by Walsh as "any pharmaceutical product used for a therapeutic or in vivo di‐ agnostic purpose, which is produced in full or in part by either traditional or modern bio‐

On comparing the definitions for a biotechnology-derived product and a biological product, both of which should be of biological origin, it is obvious that the key element in the former

In practice, the regulatory requirements needed for a biotechnology-derived product and a biological product are methodologically different from the pharmaceutical product due to their biological essence. For example, the determination of adventitious agents such as vi‐ ruses, transmitting spongiform encephalopathy (TSE), and mycoplasma are included in most of the related guidelines and pharmacopeias for a biotechnology-derived product and

Finally, a biopharmaceutical is defined by Walsh as "A protein or nucleic acid based phar‐ maceutical substance used for therapeutic or in vivo diagnostic purposes, which is produced by means other than direct extraction from a native (non-engineered) biological source." This definition that will be used in the present chapter for biopharmaceuticals includes all pharmaceutical products produced by modern biotechnology techniques as well as nucleic acid (DNA or RNA) based pharmaceutical products for gene therapy. Hence, the overlap‐ ping area between biotechnology-derived products and biopharmaceuticals is the applica‐ tion of modern biotechnological means in their production. However, the differentiating area can be the application of traditional biotechnological means for the production of bio‐ technology-derived products. In addition, nucleic acid-based pharmaceutical products that are categorized as biopharmaceuticals are not biotechnology-derived products [6]. Figure 1 illustrates these overlapping and differentiating areas. Moreover, some examples of prod‐

**Biotechnology-derived product by:**

**Biological products Traditional technology Modern technology Biopharmaceuticals**

**Table 1.** Some examples of products related to biological, biotechnology-derived, and biopharmaceutical products.

natural sources Recombinant proteins Recombinant proteins

Nucleic acid-based pharmaceutical products for gene therapy

Monoclonal antibody produced by hybridoma technology

definition is the application of biotechnological means for production.

technological means." [5]

196 Latest Research into Quality Control

a biological product.

ucts in these categories are shown in Table1.

Blood and blood products Therapeutic proteins from

Human cells and tissues Antibiotics fully or partially

from microorganisms

The main category of biopharmaceuticals is manufactured via recombinant DNA technolo‐ gy. Indeed, recombinant DNA technologies are enabling techniques that manipulate and en‐ gineer different gene fragments and which have been introduced less than 50 years ago by the revolutionary invention of Polymerase Chain Reaction (PCR) by Kary Mullis [7].

DNA and RNA extraction from different cell types, cutting DNA fragments using restriction endonucleases, joining DNA fragments by DNA ligases, PCR to amplify gene fragments, cloning of the gene fragments into different vectors, introduction of recombinant constructs into proper hosts, protein expression, extraction, and purification are some of the most widely used means in recombinant protein production.

Figure 2 schematically represents the summarized process of production of a recombinant protein. As can be seen from the chart, first, the gene of interest should be isolated and am‐ plified from the original cell. According to the type of the cell, it can be done through direct total DNA extraction followed by a PCR using proper primers to obtain the gene in prokar‐ yotes. On the other hand, in eukaryotes, due to the existence of introns and some modifica‐ tions that occur in the transcribed mRNA, the process is considerably complicated. Introns are non-coding sequences which are removed after transcription versus coding sequences that are called *exons*. In addition, mRNA is more modified by the addition of a methylated guanine (CAP) on its 5'end and a poly-adenine tail on its 3'end. After these modifications, mature mRNA is exported to the cytoplasm in order to start the translation process. Conse‐ quently, to obtain a gene of interest in eukaryotes, the mature mRNA should be extracted from the cell, and the complementary DNA should be synthesized followed by amplifica‐ tion of the gene by PCR using proper primers. However, in both cases (prokaryotes and eu‐ karyotes), the short genes can be obtained by a solid-phase synthesis process.

fragment that can be inserted into them. Ultimately, the new recombinant construct should be introduced into an expression system for production. Different classes of expression sys‐ tems with their certain merits and disadvantages are available and range from cell systems such as bacterial, animal, fungal, and yeast cells to transgenic systems such as transgenic

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Each specific antibody is secreted by a specific B cell and could recognize a specific region on the antigen that is called *epitope*. Each antibody-secreting B cell could be used as a source of an antibody of interest if it is isolated and cultured in vitro. Nevertheless, B cells are not considered a satisfactory source, as they are not immortalized and cannot survive for a long time. The main approach for overcoming this problem is cell hybridization, which includes the fusion of antibody-secreting B cells with tumor cells (such as mouse myeloma cells) pro‐ liferating ever more. The classical hybridoma technology was first introduced by Georges Kohler and Cesar Milstein in the mid 1970s for the generation of immortalized hybridoma cells that could grow in cell culture for a long time and produce the desired monoclonal an‐ tibodies [9, 10]. The basic process (Figure 3) includes the immunization of a mouse with the desired antigen. The mouse was then sacrificed, and B lymphocytes secreting antibodies that were selective for the specific epitope on the antigen were isolated from the spleen. The spleen is considered the most ready source for antigen-specific lymphocytes that provides access to a large number of antibody-secreting cells [1]. The isolated B cells were subse‐ quently fused with immortal mouse myeloma cells. The resultant hybridoma cells were then separated from the unfused cells by culturing in specific cell culture media. The cell culture media for the hybridoma growth and production of monoclonal antibodies have been re‐

In general, for the successful fusion of hybridomas, the cells are grown in HAT selection medi‐ um. The selection medium is called *HAT*, as it has Hypoxanthine, Aminopterin, and Thymi‐ dine. This is because a mutation in either the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or the thymidine kinase (TK) gene of the cells would cause their death in the HAT medium. Generally, a TK-deficient cell (TK-negative mutant) is resistant to bromodeoxyuridine (BrdU), and an HGPRT-deficient cell (HGPRT-negative mu‐ tant) is resistant to 6-thioguanine (6-TG) and 8-azaguanine. Hence, in order to make myeloma cells sensitive to HAT (unable to grow in HAT media), they are treated with one of these drugs

Normal cells can synthesize the required nucleotides in two pathways: (1) the main one or de novo biosynthetic pathway, and (2) the alternative one or the salvage pathway (when the

Aminopterin (a folic acid analog that inhibits dihydrofolate reductase) blocks the activation of tetrahydrofolate, which is required for the synthesis of nucleotides via the de novo syn‐ thetic pathway, and, therefore, the main pathway is blocked. Thus, in aminopterin-treated

plants and animals [7, 8].

*2.1.2. Monoclonal antibody production*

*<IP_ADDRESS>. Classical hybridoma technology*

viewed in detail by Bols et al. [11].

before their passage to HAT media.

main pathway is blocked).

**Figure 2.** Schematic representation of recombinant protein production process

Based on their sizes, the obtained genes can now be introduced into a proper vector for maintenance, replication, or expression purposes. Plasmids, cosmids, and bacteriophages are the most important cloning vectors that are classified according to the size of the DNA fragment that can be inserted into them. Ultimately, the new recombinant construct should be introduced into an expression system for production. Different classes of expression sys‐ tems with their certain merits and disadvantages are available and range from cell systems such as bacterial, animal, fungal, and yeast cells to transgenic systems such as transgenic plants and animals [7, 8].

#### *2.1.2. Monoclonal antibody production*

that are called *exons*. In addition, mRNA is more modified by the addition of a methylated guanine (CAP) on its 5'end and a poly-adenine tail on its 3'end. After these modifications, mature mRNA is exported to the cytoplasm in order to start the translation process. Conse‐ quently, to obtain a gene of interest in eukaryotes, the mature mRNA should be extracted from the cell, and the complementary DNA should be synthesized followed by amplifica‐ tion of the gene by PCR using proper primers. However, in both cases (prokaryotes and eu‐

karyotes), the short genes can be obtained by a solid-phase synthesis process.

198 Latest Research into Quality Control

**Figure 2.** Schematic representation of recombinant protein production process

Based on their sizes, the obtained genes can now be introduced into a proper vector for maintenance, replication, or expression purposes. Plasmids, cosmids, and bacteriophages are the most important cloning vectors that are classified according to the size of the DNA

#### *<IP_ADDRESS>. Classical hybridoma technology*

Each specific antibody is secreted by a specific B cell and could recognize a specific region on the antigen that is called *epitope*. Each antibody-secreting B cell could be used as a source of an antibody of interest if it is isolated and cultured in vitro. Nevertheless, B cells are not considered a satisfactory source, as they are not immortalized and cannot survive for a long time. The main approach for overcoming this problem is cell hybridization, which includes the fusion of antibody-secreting B cells with tumor cells (such as mouse myeloma cells) pro‐ liferating ever more. The classical hybridoma technology was first introduced by Georges Kohler and Cesar Milstein in the mid 1970s for the generation of immortalized hybridoma cells that could grow in cell culture for a long time and produce the desired monoclonal an‐ tibodies [9, 10]. The basic process (Figure 3) includes the immunization of a mouse with the desired antigen. The mouse was then sacrificed, and B lymphocytes secreting antibodies that were selective for the specific epitope on the antigen were isolated from the spleen. The spleen is considered the most ready source for antigen-specific lymphocytes that provides access to a large number of antibody-secreting cells [1]. The isolated B cells were subse‐ quently fused with immortal mouse myeloma cells. The resultant hybridoma cells were then separated from the unfused cells by culturing in specific cell culture media. The cell culture media for the hybridoma growth and production of monoclonal antibodies have been re‐ viewed in detail by Bols et al. [11].

In general, for the successful fusion of hybridomas, the cells are grown in HAT selection medi‐ um. The selection medium is called *HAT*, as it has Hypoxanthine, Aminopterin, and Thymi‐ dine. This is because a mutation in either the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or the thymidine kinase (TK) gene of the cells would cause their death in the HAT medium. Generally, a TK-deficient cell (TK-negative mutant) is resistant to bromodeoxyuridine (BrdU), and an HGPRT-deficient cell (HGPRT-negative mu‐ tant) is resistant to 6-thioguanine (6-TG) and 8-azaguanine. Hence, in order to make myeloma cells sensitive to HAT (unable to grow in HAT media), they are treated with one of these drugs before their passage to HAT media.

Normal cells can synthesize the required nucleotides in two pathways: (1) the main one or de novo biosynthetic pathway, and (2) the alternative one or the salvage pathway (when the main pathway is blocked).

Aminopterin (a folic acid analog that inhibits dihydrofolate reductase) blocks the activation of tetrahydrofolate, which is required for the synthesis of nucleotides via the de novo syn‐ thetic pathway, and, therefore, the main pathway is blocked. Thus, in aminopterin-treated cells (HGPRT+ and TK+ ), the synthesis of nucleotides shifts to the salvage pathway only if hypoxanthine and thymidine are supplied in the medium. HGPRT and TK, the two en‐ zymes, are required for the salvage pathway, and they catalyze the synthesis of purine and thymidylate from hypoxanthine and thymidine substrates, respectively.

**5.** When in vitro methods result in monoclonal antibodies that are glycosylated at posi‐ tions different from those harvested from mouse ascites, they affect antigen-binding ca‐

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Taken together, the cell culture technique is a method of choice for large-scale monoclonal antibody production due to the simplicity of the cell culture and financial considerations

**Figure 3.** The diagram of the monoclonal antibody production via classical hybridoma technology

In 1986, about 10 years after the conception of monoclonal antibody technology, Orthoclone OKT3 was approved by the FDA for use in patients with acute rejection of a transplanted kidney [14]. Unfortunately, early clinical applications of murine monoclonal antibodies were disappointing. This was due to the fact that monoclonal antibodies produced via the classi‐ cal method are of murine origin and are, therefore, immunogenic to human subjects. In gen‐ eral, patients receiving an antibody exhibit HAMA responses (human anti-mouse antibodies) within two weeks. Multiple infusions of murine monoclonal antibodies signifi‐ cantly enhance the HAMA reactions [6]. In addition, the immune system eliminates the murine monoclonal antibody molecule. Thus, murine monoclonal antibodies demonstrate short serum half lives after administration to humans. Furthermore, the other main difficul‐ ty related to murine monoclonal antibodies is the poor recognition of the Fc region by hu‐

Thus, new strategies that are used for producing humanized mouse antibodies that are less immunogenic have been discovered. The first strategy includes the production of functional specific recombinant IgG molecules consisting of mouse variable regions and human con‐ stant regions; these are known as chimeric antibodies. Taken together, in the chimeric anti‐ body, 8 out of 12 domains are of human origin (constant regions of the heavy and light chains) (CH and CL) [15]. Chimeric antibodies exhibit reduced HAMA responses compared

pacity as well as biological functions [12].

without ethical concerns that are related to animal use.

*<IP_ADDRESS>. Chimeric and humanized antibodies*

man effector systems of complement and Fc receptors.

Since unfused myeloma cells lack HGPRT or TK, they cannot use the salvage pathway. Thus, the unfused myeloma cells get killed in the HAT medium, as both biosynthetic path‐ ways are blocked. Normal unfused B cells die in the HAT medium, as they are not immor‐ talized and cannot grow for a long time. Nevertheless, the fusion of normal B cells with the HGPRTor TKmyeloma cells allows the hybridoma cells to grow in HAT medium, as the B cells provide the necessary enzymes for growth of the hybridoma cells.

Hence, the HAT selection medium offers an ideal environment for the isolation of fused myeloma and B cells (hybridoma cells) from unfused myeloma cells and unfused B cells, as this medium allows only the hybridoma cells to survive in the culture.

The production of monoclonal antibodies could be accomplished by ascites (ascitic fluid) production (in vivo) or by cell culture (in vitro) methods. In the in vivo method, hybridoma cells are injected intraperitoneally into mice. The peritoneum serves as a growth chamber for the injected cells. These cells could secrete a high-titered solution of desired antibodies as they grow in the cavity. Finally, the produced antibodies are extracted from the ascitic fluid accumulated in the peritoneal cavity [6]. The antibody concentrations typically range be‐ tween 1 and 15 mg/ml. The in vivo method offers a very high concentration of monoclonal antibody that often does not need more concentration procedures. Nevertheless, monoclonal antibodies produced by this technique may be contaminated by considerable levels of mouse proteins and other contaminants that might require more complicated, subsequent downstream purifications. The other disadvantage of the ascites production is related to ani‐ mal welfare issues, as these could cause distress in mice.

Currently, more than 90% of monoclonal antibodies are produced by in vitro techniques [12] that use large-scale manufacturing plants containing several 10,000-L or larger culture bio‐ reactors [13]. The in vitro method of monoclonal antibody production decreases the use of mice and also avoids the need for experienced personnel for animal handling. Regardless of the privileges and importance of the in vitro methods of antibody production, there are some situations in which this method is not applicable; for instance:

**5.** When in vitro methods result in monoclonal antibodies that are glycosylated at posi‐ tions different from those harvested from mouse ascites, they affect antigen-binding ca‐ pacity as well as biological functions [12].

Taken together, the cell culture technique is a method of choice for large-scale monoclonal antibody production due to the simplicity of the cell culture and financial considerations without ethical concerns that are related to animal use.

**Figure 3.** The diagram of the monoclonal antibody production via classical hybridoma technology

#### *<IP_ADDRESS>. Chimeric and humanized antibodies*

cells (HGPRT+

200 Latest Research into Quality Control

HGPRT-

or TK-

activity.

valuable hybridoma.

and TK+

), the synthesis of nucleotides shifts to the salvage pathway only if

hypoxanthine and thymidine are supplied in the medium. HGPRT and TK, the two en‐ zymes, are required for the salvage pathway, and they catalyze the synthesis of purine and

Since unfused myeloma cells lack HGPRT or TK, they cannot use the salvage pathway. Thus, the unfused myeloma cells get killed in the HAT medium, as both biosynthetic path‐ ways are blocked. Normal unfused B cells die in the HAT medium, as they are not immor‐ talized and cannot grow for a long time. Nevertheless, the fusion of normal B cells with the

Hence, the HAT selection medium offers an ideal environment for the isolation of fused myeloma and B cells (hybridoma cells) from unfused myeloma cells and unfused B cells, as

The production of monoclonal antibodies could be accomplished by ascites (ascitic fluid) production (in vivo) or by cell culture (in vitro) methods. In the in vivo method, hybridoma cells are injected intraperitoneally into mice. The peritoneum serves as a growth chamber for the injected cells. These cells could secrete a high-titered solution of desired antibodies as they grow in the cavity. Finally, the produced antibodies are extracted from the ascitic fluid accumulated in the peritoneal cavity [6]. The antibody concentrations typically range be‐ tween 1 and 15 mg/ml. The in vivo method offers a very high concentration of monoclonal antibody that often does not need more concentration procedures. Nevertheless, monoclonal antibodies produced by this technique may be contaminated by considerable levels of mouse proteins and other contaminants that might require more complicated, subsequent downstream purifications. The other disadvantage of the ascites production is related to ani‐

Currently, more than 90% of monoclonal antibodies are produced by in vitro techniques [12] that use large-scale manufacturing plants containing several 10,000-L or larger culture bio‐ reactors [13]. The in vitro method of monoclonal antibody production decreases the use of mice and also avoids the need for experienced personnel for animal handling. Regardless of the privileges and importance of the in vitro methods of antibody production, there are

**2.** Downstream purification methods cause protein denaturation or decreased antibody

**4.** When hybridoma cells are contaminated with infectious agents (such as yeasts or fun‐ gi), the cells must often be passed through mice. Since removal of the organisms cannot be accomplished by current antimicrobial drugs, thus the in vivo method may save a

myeloma cells allows the hybridoma cells to grow in HAT medium, as the B

thymidylate from hypoxanthine and thymidine substrates, respectively.

cells provide the necessary enzymes for growth of the hybridoma cells.

this medium allows only the hybridoma cells to survive in the culture.

mal welfare issues, as these could cause distress in mice.

some situations in which this method is not applicable; for instance:

**3.** The cell line cannot maintain the production of monoclonal antibodies.

**1.** Hybridoma cells do not adapt well to in vitro conditions.

In 1986, about 10 years after the conception of monoclonal antibody technology, Orthoclone OKT3 was approved by the FDA for use in patients with acute rejection of a transplanted kidney [14]. Unfortunately, early clinical applications of murine monoclonal antibodies were disappointing. This was due to the fact that monoclonal antibodies produced via the classi‐ cal method are of murine origin and are, therefore, immunogenic to human subjects. In gen‐ eral, patients receiving an antibody exhibit HAMA responses (human anti-mouse antibodies) within two weeks. Multiple infusions of murine monoclonal antibodies signifi‐ cantly enhance the HAMA reactions [6]. In addition, the immune system eliminates the murine monoclonal antibody molecule. Thus, murine monoclonal antibodies demonstrate short serum half lives after administration to humans. Furthermore, the other main difficul‐ ty related to murine monoclonal antibodies is the poor recognition of the Fc region by hu‐ man effector systems of complement and Fc receptors.

Thus, new strategies that are used for producing humanized mouse antibodies that are less immunogenic have been discovered. The first strategy includes the production of functional specific recombinant IgG molecules consisting of mouse variable regions and human con‐ stant regions; these are known as chimeric antibodies. Taken together, in the chimeric anti‐ body, 8 out of 12 domains are of human origin (constant regions of the heavy and light chains) (CH and CL) [15]. Chimeric antibodies exhibit reduced HAMA responses compared with mouse antibodies, but the affinity and the selectivity are the same. Furthermore, since the Fc region contains human sequences, the activation of Fc-mediated immune effector functions is allowed.

**2.2. Mass production of a recombinant product**

**Figure 5.** Mass production of a recombinant product.

Figure 5.

Mass production of recombinant products can be achieved in a process that is divided into two main sections called *upstream* and *downstream* processing, as schematically depicted in

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The first step, the upstream processing step, is the mass production of a recombinant protein via the fermentation process. The original recombinant system that is used for the expres‐ sion of the recombinant protein (i.e. in the form of a recombinant plasmid maintained in a suitable host cell) forms the cell deposit in a cell banking system. There are two levels of cell deposits in the cell banking system: The first line is called *master cell bank*, which is prepared

To further minimize the antigenicity of murine antibodies, humanized antibodies were devel‐ oped. For their generation, hyper-variable complementarity-determining regions (CDRs) of the specific murine antibody are transferred to a fully human framework. In comparison with the mouse antibodies, humanized antibodies suggest a lower occurrence of HAMA responses.

Further efforts have been invested in the development of technologies that generate fully hu‐ man monoclonal antibodies. One of the approaches entails the development of transgenic mice, in which a repertoire of human immunoglobulin germline gene segments is inserted into the mouse genome. After the immunization of these mice, they produce fully human antibodies, which can subsequently be separated with the classical hybridoma technology [15].

Figure 4 illustrates the schematic structures of mouse, chimeric, humanized, and human antibodies.

**Figure 4.** The structure of mouse (shown in red color), chimeric, humanized (shown in black color), and human anti‐ bodies. Chimeric antibodies comprise mouse variable regions and human constant regions. Humanized antibodies consist of murine hyper-variable complementarity-determining regions (CDRs) that are grafted to fully human frame‐ work.

#### **2.2. Mass production of a recombinant product**

with mouse antibodies, but the affinity and the selectivity are the same. Furthermore, since the Fc region contains human sequences, the activation of Fc-mediated immune effector

To further minimize the antigenicity of murine antibodies, humanized antibodies were devel‐ oped. For their generation, hyper-variable complementarity-determining regions (CDRs) of the specific murine antibody are transferred to a fully human framework. In comparison with the mouse antibodies, humanized antibodies suggest a lower occurrence of HAMA responses.

Further efforts have been invested in the development of technologies that generate fully hu‐ man monoclonal antibodies. One of the approaches entails the development of transgenic mice, in which a repertoire of human immunoglobulin germline gene segments is inserted into the mouse genome. After the immunization of these mice, they produce fully human antibodies,

Figure 4 illustrates the schematic structures of mouse, chimeric, humanized, and human

**Figure 4.** The structure of mouse (shown in red color), chimeric, humanized (shown in black color), and human anti‐ bodies. Chimeric antibodies comprise mouse variable regions and human constant regions. Humanized antibodies consist of murine hyper-variable complementarity-determining regions (CDRs) that are grafted to fully human frame‐

which can subsequently be separated with the classical hybridoma technology [15].

functions is allowed.

202 Latest Research into Quality Control

antibodies.

work.

Mass production of recombinant products can be achieved in a process that is divided into two main sections called *upstream* and *downstream* processing, as schematically depicted in Figure 5.

**Figure 5.** Mass production of a recombinant product.

The first step, the upstream processing step, is the mass production of a recombinant protein via the fermentation process. The original recombinant system that is used for the expres‐ sion of the recombinant protein (i.e. in the form of a recombinant plasmid maintained in a suitable host cell) forms the cell deposit in a cell banking system. There are two levels of cell deposits in the cell banking system: The first line is called *master cell bank*, which is prepared directly from a culture of the original recombinant system that includes several hundred stored ampoules.

Furthermore, high-temperature, short-time treatment strategies are sometimes employed for the elimination of biological contaminants from small solutes such as vitamins and amino

Another important raw material that is used in the production of any pharmaceutical prod‐ uct, including biopharmaceuticals, is water, which can be considered an important source for contamination, with water-borne bacteria such as Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacter spp., and Serratia spp. Water for pharmaceutical pur‐ poses is discussed in detail in the USP [17]. Due to the fact that the intended administration of biopharmaceuticals in the majority of cases is via injection, Water for Injection (WFI)

Pharmaceutical products' contamination may occur from the transformation of microorgan‐ isms from the production environment to the product. The production environment in‐

The main groups of microorganisms that are isolated from air are the spore-forming bacteria (Bacillus spp. and Clostridium spp., the non-sporing bacteria Staphylococcus spp., Streptococ‐ cus spp., and Corynebacterium spp.), the molds (Penicillium spp., Cladosporium spp., Asper‐ gillus spp., and Mucor spp.), and the yeast (Rhodotorula spp.). These contaminants may be air borne or can be initiated from process equipment or personnel [16]. Consequently, environ‐ mental monitoring programs in a production environment are essential actions. Furthermore, the critical operations in biopharmaceutical production should be performed in controlled en‐ vironments or clean rooms. A clean room is a place with high control of the entrance of parti‐ cles via the establishment of some air filters called *high-efficiency particulate air (HEPA) filters*. HEPA filters made from a microglass material with a pleated construction system provide a large surface area that efficiently filters the incoming air and generates a constant air motion. Based on the permitted quantity of viable microorganisms and particulates, various classes of

According to the *EC Guide to Good Manufacturing Practice for Medicinal Products* (EC GGMP), four grades for clean rooms are available, such as grade A, B, C, or D, based on the number

> **Maximum permitted CFU of viable microorganisms/m** <sup>3</sup> **in air sample**

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which is sterile and apyrogen is routinely used in this area.

cludes air, surfaces, instruments, equipments, and personnel.

clean rooms can be established using HEPA filters with required efficiencies.

**particles/m** <sup>3</sup>

A 3500 <1 B 3500 10 C 350000 100 D 3500000 200

**Table 2.** Clean room grades according to the number of viable microorganisms and the number of particulates

of viable microorganisms and particulates (Table 2).

**Clean room grade Maximum permitted number of**

acids [16].

**3.2. Production environment**

The second line that is referred to as the *working cell bank* is produced from a single master cell bank ampoule. Each ampoule from the working cell bank is thawed and used to seed the fermentor for the production of a batch. Obviously, the fermentation process for various recombinant systems, such as bacterial fermenting systems or animal cell systems, is quite different and needs its own certain requirements.

On the other hand, *downstream processing*, which is the next step, refers to the purification of the mass produced protein. The first step in downstream processing is the initial extraction and concentration of the product, which depends on the situation of the expressed protein; that is, it should be extracted from the pellet cells for intracellular proteins or from the su‐ pernatant for extracellular proteins after centrifugation.

The second stage entails (1) several chromatographic steps that complete the purification of the product; (2) the potency test; (3) the addition of suitable excipients; (4) sterilization; (5) filling of the product in its final form (liquid or solid) into the final container before sealing; and labeling [6].

#### **3. Sources of biological contamination of biopharmaceuticals**

In line with conventional pharmaceutical products, the main sources of biological contami‐ nation in biopharmaceuticals can be related to raw materials and the production environ‐ ment. Indeed, the biological contaminant content of any pharmaceutical product is a representative of their starting materials and the production environment flora.

#### **3.1. Raw materials**

Animal origin materials, such as cell culture media, sera, and supplements that are exten‐ sively used in biopharmaceutical production, are of high contamination risk. These materi‐ als can be considered the main source for the contamination of biopharmaceuticals with adventitious agents such as TSEs, viruses, and mycoplasmas. Therefore, they should be sup‐ plied from reliable resources, and special attention should be paid to their quality control procedure. It should be ensured that all raw materials, especially those of high risk, gain quality specifications for current good manufacturing practice.

Standard methods for sterilization of cell culture media, sera, and supplements should be established according to the properties of the materials. Due to the heat-labile nature of the majority of materials used in biopharmaceutical production, autoclaving is usually replaced with alternative strategies such as filter-sterilization or less frequently high-temperature, short-time treatment strategies. In spite of the routine filter-sterilization procedure that uses 0.22 μm, it is usually performed with 0.1-μm membrane filters due to the risk of contamina‐ tion with adventitious agents.

Furthermore, high-temperature, short-time treatment strategies are sometimes employed for the elimination of biological contaminants from small solutes such as vitamins and amino acids [16].

Another important raw material that is used in the production of any pharmaceutical prod‐ uct, including biopharmaceuticals, is water, which can be considered an important source for contamination, with water-borne bacteria such as Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacter spp., and Serratia spp. Water for pharmaceutical pur‐ poses is discussed in detail in the USP [17]. Due to the fact that the intended administration of biopharmaceuticals in the majority of cases is via injection, Water for Injection (WFI) which is sterile and apyrogen is routinely used in this area.

#### **3.2. Production environment**

directly from a culture of the original recombinant system that includes several hundred

The second line that is referred to as the *working cell bank* is produced from a single master cell bank ampoule. Each ampoule from the working cell bank is thawed and used to seed the fermentor for the production of a batch. Obviously, the fermentation process for various recombinant systems, such as bacterial fermenting systems or animal cell systems, is quite

On the other hand, *downstream processing*, which is the next step, refers to the purification of the mass produced protein. The first step in downstream processing is the initial extraction and concentration of the product, which depends on the situation of the expressed protein; that is, it should be extracted from the pellet cells for intracellular proteins or from the su‐

The second stage entails (1) several chromatographic steps that complete the purification of the product; (2) the potency test; (3) the addition of suitable excipients; (4) sterilization; (5) filling of the product in its final form (liquid or solid) into the final container before sealing;

In line with conventional pharmaceutical products, the main sources of biological contami‐ nation in biopharmaceuticals can be related to raw materials and the production environ‐ ment. Indeed, the biological contaminant content of any pharmaceutical product is a

Animal origin materials, such as cell culture media, sera, and supplements that are exten‐ sively used in biopharmaceutical production, are of high contamination risk. These materi‐ als can be considered the main source for the contamination of biopharmaceuticals with adventitious agents such as TSEs, viruses, and mycoplasmas. Therefore, they should be sup‐ plied from reliable resources, and special attention should be paid to their quality control procedure. It should be ensured that all raw materials, especially those of high risk, gain

Standard methods for sterilization of cell culture media, sera, and supplements should be established according to the properties of the materials. Due to the heat-labile nature of the majority of materials used in biopharmaceutical production, autoclaving is usually replaced with alternative strategies such as filter-sterilization or less frequently high-temperature, short-time treatment strategies. In spite of the routine filter-sterilization procedure that uses 0.22 μm, it is usually performed with 0.1-μm membrane filters due to the risk of contamina‐

**3. Sources of biological contamination of biopharmaceuticals**

representative of their starting materials and the production environment flora.

quality specifications for current good manufacturing practice.

stored ampoules.

204 Latest Research into Quality Control

and labeling [6].

**3.1. Raw materials**

tion with adventitious agents.

different and needs its own certain requirements.

pernatant for extracellular proteins after centrifugation.

Pharmaceutical products' contamination may occur from the transformation of microorgan‐ isms from the production environment to the product. The production environment in‐ cludes air, surfaces, instruments, equipments, and personnel.