Detailed information concerning the composition of the raw materials employed in the production of biologics is important for the efficient control and optimization of bioprocesses.  We demonstrate the application of electrophoresis microchips with capacitively-coupled contactless conductivity detection (C4D) to the analysis of wa-ter-soluble vitamins and metal cations in raw material solutions that are subse-quently fed into bioreactors for the production of biologics

Benito-Lopez, Fernando

Diamond, Dermot

Garcia-Cordero, Jose L.

Ricco, Antonio J.

Vázquez, Mercedes

English

Irish Universities

Analysis of biopharma raw materials by electrophoresis microchips with contactless conductivity detection

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ANALYSIS OF BIOPHARMA RAW MATERIALS BY ELECTROPHORESIS MICROCHIPS WITH CONTACTLESS CONDUCTIVITY DETECTION Mercedes Vázquez1, Fernando Benito-López1, José L. García-Cordero2, Antonio J. Ricco2 and Dermot Diamond1,2 1 Centre for Bioanalytical Sciences and 2Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin 9, IRELAND  ABSTRACT Detailed information concerning the composition of the raw materials employed in the production of biologics is important for the efficient control and optimization of bioprocesses.  We demonstrate the application of electrophoresis microchips with capacitively-coupled contactless conductivity detection (C4D) to the analysis of wa-ter-soluble vitamins and metal cations in raw material solutions that are subse-quently fed into bioreactors for the production of biologics.   KEYWORDS: Electrophoresis microchip, Contactless conductivity detection, Bio-pharma raw materials, Vitamins, Metal cations  INTRODUCTION The analysis of vitamins in industry is typically performed using high-performance liquid chromatography (HPLC) with single-wavelength UV detection (or a photodiode array, PDA, spectrometer), whereas for the analysis of metal cations, inductively-coupled plasma (ICP) methods are used with various detection systems such as atomic emission spectrometry (AES).  Compared to those methods, the use of electrophoresis microchips represents a cost-effective and promising tool for application in the analysis of raw materials in biologics: analysis times can be re-duced to seconds and high separation efficiencies can be achieved using extremely small volume samples (a few µL), and minimal reagent consumption/waste genera-tion.  Additionally, the use of C4D offers a rather simple and sensitive method for detection of ionic species with no need for sample derivatization.  Though the analy-sis of real samples with electrophoresis microchips remains challenging, a signifi-cant number of publications dealing with real food samples has been published in recent years [1-2].  However, to the best of our knowledge, this is the first report of the analysis of vitamins and metal ions in real biopharma raw materials using elec-trophoresis microchips.  EXPERIMENTAL A commercially available C4D detector (TraceDec, Austria) was integrated with a hybrid PDMS/glass microchip and copper sensing electrodes (Figure 1).  The cop-per electrodes were fabricated in-house using a commercially available printed cir-cuit board (PCB) (Figure 1D).  This fabrication procedure is simple, inexpensive and enables the construction of very robust sensing electrodes that can be reused multi-ple times.   Figure 1.  System setup (A) and layout of the hybrid PDMS/glass microchip and the C4D electrodes (B).  Process schemes for the fabrication of the master used in the production of the PDMS layer with embedded microchannels (channel dimen-sions: 45 µm x 50 µm) (C), and the fabrication of the C4D electrodes (D).  RESULTS AND DISCUSSION Separation of six metal cations in commercially available biopharma raw materials is demonstrated in Figure 2.  Commercially available chemically-defined Chinese ham-ster ovary (CD-CHO) media samples were analyzed after sample dilution of 1:10,000.    Figure 2.  Electropherogram for 6 metals in a commercial CD-CHO sample.  Electropherogram obtained with a standard solution (50 µM each) is shown for comparison.  Run buffer: 10 mM MES/His (pH 6.0).  Effective length: 4.0 cm.  Injec-tion voltage: 0.8 kV (2 s).  Separation voltage: 1.2 kV.  MES= 2-(N-Morpholino)ethanesulfonic acid, His = Histidine.  A solid-phase extraction method (SPE) was also developed for the extraction of water-soluble vitamins from CD-CHO media by using a SPE cartridge based on a polymeric sorbent.  The vitamins retained in the SPE cartridge were eluted with a mixture of 30 % acetonitrile in water after a washing step.  Eluting solvents were then evaporated and the resulting sample was reconstituted in water.  The analysis of four water-soluble vitamins in standard mixtures is presented in Figure 3A before and after spiking the standards with vitamin B12.  Application of the SPE method and the separation method developed for vitamins to a commercial CD-CHO sample is presented in Figure 3B.  Prior to analysis, the vitamin extract, reconstituted in wa-ter after SPE, was further diluted 1:10.   Figure 3.  Electropherograms showing the separation of vitamins in a standard solution (10 µM each) before and after spiking with B12 (A) and in a commercial CD-CHO sample (B).  Run buffer: 30 mM lactic acid (pH 2.6).  Effective length: 3.7 cm.  Injection potential:0.8 kV (1.5 s).  Separation potential: 1.2 kV.  CONCLUSIONS The application of electrophoresis microchips with C4D detection to the ultra-fast analysis of water-soluble vitamins and metals in raw materials used for the produc-tion of biologics was demonstrated.  Sucessful separation of six metal cations in commercial CD-CHO samples was achieved in less than 70 s.  Likewise, separation of four water-soluble vitamins was achieved in less than 40 s.  ACKNOWLEDGEMENTS Financial support from the Industrial Development Authority, Ireland (IDA pro-ject #116294), Irish Research Council for Science, Engineering, and Technology (Fellowship No. 2089), Science Foundation Ireland (Grant No. 05/CE3/B754), and Bristol-Myers Squibb is gratefully acknowledged.  REFERENCES [1] A.  Escarpa, M.C.  González, M.A.  López Gil, A.G.  Crevillén, M.  Hervás, M.  García, Microchips for CE: Breakthroughs in real-world food analysis,  Electrophoresis, 29, pp.  4852-4861, (2008). [2] W.S.  Law, P.  Kubáň, J.H.  Zhao, S.F.Y.  Li, P.C.  Hauser, Determination of vitamin C and preservatives in beverages by conventional capillary electropho-resis and microchip electrophoresis with capacitively coupled contactless con-ductivity detection,  Electrophoresis, 26, pp.  4648-4655, (2005). 

Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection,

Microchips for CE: Breakthroughs in real-world food analysis,

Name not available

ANALYSIS OF BIOPHARMA RAW MATERIALS BY ELECTROPHORESIS MICORCHIPS WITH CONTACTLESS CONDUCTIVITY DETECTIONMercedes Vazquez1, Fernando Benito-López1, José L. García-Cordero2, Antonio J. Ricco2 and Dermot Diamond1,21Centre for Bioanalytical Sciences, 2Biomedical Diagnostics Institute, National Centre for Sensor Research,Dublin City University, Dublin 9, IrelandE-mail: mercedes.vazquez@dcu.ie, fernando.lopez@dcu.ieDetailed information concerning the composition of the raw materials employed in the production of biologics is important for the efficient control and optimization of bioprocesses. Vitamins are present at very low concentrations in biopharma raw materials and are usually determined by HPLC with a UV detector or a photodiode array spectrometer (PDA). For the analysis of metal cations, inductively-coupled plasma (ICP) methods are used with various detection systems such as atomic emission spectrometry (AES). Microchip-based electrophoresis represents a cost-effective and promising tool for application in the analysis of raw materials in biologics since analysis times can be reduced to seconds and high separation efficiencies can be achieved using extremely low volume samples (a few µL), minimal reagent consumption and waste generation, low cost/disposability, portability and ease of mass-production. additionally, Capacitively Coupled Contactless Conductivity Detection (C4D) offers a rather simple and yet sensitive method for detection of ionic species with no need for sample derivatization. There is no physical contact of the detection electrodes with the electrolyte solution. Therefore, the integration of this detection mode within the analytical system is rather simple. Though the analysis of real samples with electrophoresis microchips remains challenging, a significant number of publications dealing with real food samples has been published in recent years [1-2].  However, to the best of our knowledge, this is the first report of the analysis of vitamins and metal ions in real biopharma raw materials using electrophoresis microchips.INTRODUCTIONSET-UP ACKNOWLEDGMENTSFinancial support from the Industrial Development Authority Ireland (IDA, project # 116294), Irish Research Council for Science, Engineering, and Technology (IRCSET, fellowship No. 2089), Science Foundation Ireland (SFI, grants 05/CE3/B754 & 07/CE/I1147), and BMS is gratefully acknowledged.Power supply Shielding boxC D detector4C D sensor4 MicrochipPDMS layerGlass layerPCB substrateC D electrodes4Figure 1. Picture showing the system setup (left). Layout of the hybrid PDMS/glass microchip and the C4D electrodes (right). Channel dimensions: 45 µm x 50 µm.Figure 2. Scheme showing the fabrication of the master for the production of the PDMS layer with embedded microchannels (A) and the fabrication of the C4D electrodes (B).(A) (B)SPE cartridge (Polymeric sorbent)1. Loading with 150 l sample2. Wash with 1ml water3. Elution with 500 l 30% ACN4. Evaporation & reconstitution in 150 l waterExtractVitaminsInterferents0 10 20 30 40 50 60 70-10-8-6-4-20246K+Ba2+Na+Li+ Cd2+ Zn2+Li+InjectionZn2+Cd2+Na+Ba2+K+   C4 D Signal (mV)Time (s) Standard (50 M) CD CHO sample (diluted 1:10,000)0 10 20 30 40 501600160516101615162016251630InjectionB12BiotinPantothenic AcidB6  C4D signal (mV)Time (s) Standard CD CHO sample (diluted 1:10)0 10 20 30 40 5015881592159616001604160816121616InjectionB12BiotinPantothenic AcidB6  C4 D Signal (mV)Time (s) Standard Standard spiked with B12Figure 3. Electropherogram for 6 metals in a standard solution (50 µM each) and in a commercial CD-CHO sample. Run buffer: 10 mM MES/His (pH 6.0).  Effective length: 4.0 cm.  Injection voltage: 0.8 kV (2 s). Separation voltage: 1.2 kV. C4D parameters: frequency,300 kHz; excitation voltage, 20 Vpeak-to-peak. Figure 4. Solid Phase Extraction (SPE) method developed for extraction of water-soluble vitamins from  biopharma raw-material samples.Figure 5. Electropherograms showing the separation of vitamins in a standard solution (10 µM each) before and after spiking with B12 (A) and in a commercial CD-CHO sample (B).  Run buffer: 30 mM lactic acid (pH 2.6).  Effective length: 3.7 cm.  Injection potential:0.8 kV (1.5 s).  Separation potential: 1.2 kV. C4D parameters: frequency, 300 kHz; excitation voltage, 20 Vpeak-to-peak.(A) (B)CONCLUSIONSREFERENCES[1] A. Escarpa, M.C. González, M.A. López Gil, A.G. Crevillén, M. Hervás, M. García, Microchips for CE: Breakthroughs in real-world food analysis,Electrophoresis, 29, pp.  4852-4861, (2008).[2] W.S. Law, P. Kubáň, J.H. Zhao, S.F.Y. Li, P.C. Hauser, Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection, Electrophoresis, 26, pp.  4648-4655, (2005).RESULTSMICROHIP & ELECTRODE FABRICATIONThe application of electrophoresis microchips with C4D detection to the ultra-fast analysis of water-soluble vitamins and metals in raw materials used for the production of biologics was demonstrated. Successful separation of six metal cations in commercial CD-CHO samples, with no sample prep, was achieved in less than 70 s.  Likewise, separation of four water-soluble vitamins was achieved in less than 40 s after SPE.

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Analysis of biopharma raw materials by electrophoresis microchips with contactless conductivity detection

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