Physico-chemical analysis
VelaLabs offers analysis according to the European Pharmacopoeia such as measuring of (sub)visible particles, coloration, turbidity and protein content.
Impurity identification and content determination via High Performance Liquid Chromatography.
Meeting your needs is our priority?
Physico-chemical analysis
Physiochemical analysis is an essential step in verifying medication integrity before and after release on the market. With physiochemical tests, confirmation of purity and identity of drugs, detection of impurities and compliance with health regulations are possible.
At VelaLabs, we offer analysis of your product according to the European Pharmacopeia as well as product-specific method setup, verification, qualification and validation.
HPLC capabilities
At VelaLabs, we are equipped to analyze your product for purity, impurity, stability and component quantification with our (U)HPLC systems. To do this, we offer assays that utilize detection with ultraviolet (UV/VWD), evaporative light scattering (ELSD) and refractive index (RID) changes.
VelaLabs offers analysis of your product with already established assays such as RPC, SEC, IEX, HIC or HILIC.
We use cutting edge technology such as Vanquish HPLCs (Thermo Scientific) and Agilent 1260 HPLCs in combination with ELSD and RID sourced from Agilent as well.
HPLC Services
There is a variety of HPLC method established at VelaLabs that will ensure product reliability and safety. Qualities we can test for:
- Purity and Impurity of Proteins (CEX, SEC, IEX and RPC)
- Excipient analysis with RID and ELSD
- Quantification of excipients with ELSD (HILIC)
- Identity by peptide mapping
Furthermore, we offer development, qualification and validation of methods that are uniquely suited to your product. During the process we investigate following parameters to ensure assay suitability and performance:
- Resolution Optimization
- Gradient selection
- Column selection
- Consideration of different HPLC systems
- Analyte preparation
- Design of Experiment (DoE)
Working principles HPLC
With (ultra) high performance liquid chromatography ((U)HPLC) it is possible to separate, identify and quantify components in products. It is indispensable in the pharmaceutical industry to monitor drug quality from development to production and approval.
Key factors that allow the HPLC to detect and separate compounds are the stationary phase and the mobile phase. The stationary phase is usually contained in a column and consists of chemically modified particles that allow the separation of the product based on the desired attribute (i.e. Molecule size or charge). The mobile phase is the solution delivered through the columns together with the analyte by a pump.
Separation of the analyte is based on its interaction with either the stationary or the mobile phase: compounds that interact stronger with the mobile phase are eluted and detected earlier than compounds that have stronger affinity to the stationary phase.
Common interactions of compounds that are investigated by HPLC assays are Hydrophobicity (with RPC), Electrostaticity (with IEX) and molecular size (with SEC).
FAQs about HPLC
When should HPLC be used, and when should GC be used?
HPLC is ideal for non-volatile, thermally unstable or high-molecular-weight compounds, whereas GC is suitable for volatile and heat-stable substances.
Find out more on our Tentamus homepage about the determination of contaminants: HPLC vs. GC
What is the duration of a HPLC analysis?
Depending on the method, the analysis itself takes between a few minutes and around an hour. Gradient methods generally take slightly longer than isocratic ones. However, the results are not available immediately. In addition, the time required for sample registration, sample preparation and evaluation must be taken into account.
What is the difference between HPLC and LC?
LC (Liquid Chromatography) ist der Oberbegriff für alle Flüssigchromatographie-Verfahren. HPLC ist eine spezielle Form davon, bei der unter hohem Druck und mit feinkörnigem Säulenmaterial gearbeitet wird, um eine höhere Trennleistung zu erzielen. HPLC bietet dadurch schnellere, präzisere und reproduzierbarere Analysen als klassische LC.
What factors influence the separation performance in HPLC?
The separation performance in HPLC is primarily influenced by the choice of stationary phase (chemistry, particle size, column length, pore size) and mobile phase (composition, polarity, pH). Flow rate, temperature and the elution mode (isocratic or gradient) also play an important role. For optimal results, all parameters must be tailored to the chemical properties of the analytes.
What is UHPLC?
Ultra-high-performance liquid chromatography (UHPLC) is used when the highest resolution and very short analysis times are required. Thanks to smaller particles (< 2 µm) and higher pressure, it separates even very similar substances more effectively than conventional HPLC. It is particularly suitable for complex samples, small sample volumes and time-critical analyses.
What is SEC-HPLC?
SEC-HPLC (Size Exclusion Chromatography) is a form of HPLC in which molecules are separated according to their size. Larger molecules pass through the pores of the stationary phase more quickly, whilst smaller ones penetrate deeper and are delayed. It is frequently used for the analysis of proteins, enzymes or polymers.
How much does an HPLC analysis cost?
The costs depend on the level of detail of the analysis, the sample matrix and the detection method used.
Get in touch with our business development team for your project!
What is the difference between an internal and an external standard?
An internal standard is added directly to the sample, whilst an external standard is measured separately; both are used to quantify the mixture of substances being analysed.
What is the capacity factor in HPLC?
The retention factor (also known as the retention time factor, k) is an important parameter in HPLC that describes how long an analyte remains in the stationary phase relative to the mobile phase. It is calculated from the difference between the retention time of the analyte (tR) and the dead time (t0, the time taken by substances not retained by the column) and then divided by t0. A high capacity factor means that the analyte interacts with the stationary phase for a long time, whilst a low value indicates rapid elution. Optimal values are usually between 1 and 10, as values that are too low may indicate insufficient separation, whilst values that are too high may indicate very long analysis times. The retention factor is independent of the flow rate and is therefore a stable parameter for method development and the comparability of analyses. It plays a central role in optimising separation performance and run time in HPLC.
Compendial Methods (according to Ph. Eur. / USP)
Compendial methods are performed on products that are in liquid form to identify and quantify chemical properties. At VelaLabs, we offer a wide range of compendial methods, performed according to Ph. Eur. and USP, including:
- pH determination (Ph. Eur. 2.2.3)
- Osmolality (Ph. Eur. 2.2.35, USP <785>)
- Sub-Visible Particles via light obscuration (Ph. Eur. 2.9.19, USP <787>, USP <788>, USP <789>, USP <1788>)
- Coloration (Ph. Eur. 2.2.2, Ph. Eur. 2.2.1, Ph. Eur. 0008, USP <631>, USP <1061>)
- Turbidity (Ph. Eur. 2.2.1, USP <855>)
- Density determination
- Endotoxin determination (Ph. Eur. 2.6.14)
- Visible Particles (Ph. Eur. 2.9.20)
- Extractable Volumes (Ph. Eur. 2.9.17)
- Concentration Determination (OD280) (Ph. Eur. 2.5.33)
- Conductivity (Ph. Eur. 2.2.38)
- TOC (Total Organic Carbon) (Ph. Eur. 2.2.44, USP <643>)
Wet chemistry methods
Gel Electrophoresis
Gel electrophoresis is an analytical method used to separate proteins or nucleic acids based on their size and charge. Depending on the assay format, separation is performed in polyacrylamide or agarose gels under an applied electric field.
For protein analysis, SDS-PAGE is one of the most commonly used techniques. Proteins are denatured and coated with sodium dodecyl sulphate (SDS), resulting in separation primarily according to molecular weight. Analyses can be performed under reducing or non-reducing conditions to evaluate disulfide bond formation, fragmentation or aggregation.
IEF-PAGE (isoelectric focusing PAGE) separates proteins according to their isoelectric point (pI) within a defined pH gradient. This technique is particularly useful for evaluating charge heterogeneity, isoform distribution and post-translational modifications of biologics.
Western blotting combines gel electrophoresis with antibody-based detection of specific proteins after transfer onto a membrane. It is commonly used for protein identification, confirmation of expression and characterization of target-specific binding.
Agarose gel electrophoresis is mainly applied for the characterization of DNA or RNA fragments. Molecules migrate through the gel matrix depending on fragment size, allowing visualization of PCR products, plasmids or nucleic acid integrity.
Practical applications include purity determination, identity testing, detection of degradation products and monitoring of process-related impurities in biologics such as antibodies, vaccines, recombinant proteins and nucleic acid-based products.
FAQs about Gel Electrophoresis
What are the benefits?
Capillaries are utilized to improve resolution and reproducibility, when compared to a standard gel electrophoresis and/or SDS-Page.
What are reducing and non-reducing conditions?
Proteins can be analysed under non-reducing conditions which means disulfide bonds are preserved, which is used for separation of non-glycosylated and intact antibody molecules as well as fragments and impurities that may be present in the sample.
Reducing conditions break down the disulfide bonds within proteins causing significant structural change. This opens the possibility to separate heavy chains and light chains of antibodies.
How are samples quantified?
Protein profiling occurs through absorbance in UV range at 220 nm (CE-SDS) and 280 nm or fluorescence (cIEF).
Capillary Electrophoresis (CE)
CE stands for capillary electrophoresis. It is used to analyze proteins based on the size or charge. Here we can differentiate between CE-SDS and cIEF.
cIEF separates molecules based on their isoelectric point (pI). A pH gradient is established within a capillary, and molecules travel until they reach their respective pI, at which a molecule has a net neutral charge.
CE-SDS separates molecules based on their size and is be performed under reducing or non-reducing conditions. Samples are denatured, bonded with sodium dodecyl sulphate and separated via electrophoresis in a capillary.
Practical applications include determination of purity, heterogeneity and/or identity of various biologics such as vaccines, antibodies, ADCs and other proteins.
We carry out the entire process using a cartridge-based imaging system (‘Maurice’) which avoids manual handling of capillaries and has integrated usage tracking.
FAQs about Capillary Electrophoresis
What is gel electrophoresis used for?
Gel electrophoresis is used to separate and analyze proteins or nucleic acids based on their physical properties. It is commonly applied for purity testing, identity confirmation and detection of impurities or degradation products.
What is the difference between SDS-PAGE and agarose gel electrophoresis?
SDS-PAGE is mainly used for protein analysis and separates molecules according to molecular weight. Agarose gel electrophoresis is primarily used for DNA or RNA fragment analysis.
Why are reducing and non-reducing conditions important in SDS-PAGE?
Reducing conditions break disulfide bonds within or between proteins, while non-reducing conditions preserve them. Comparing both conditions helps evaluate protein structure, aggregation and fragmentation.
PCR/qPCR Testing for Batch Release and Analytical Development
The polymerase-chain reaction technology is essential for biopharmaceutical development, quality control, and regulatory compliance.
At VelaLabs we offer various PCR and qPCR assays enabling analysis across a broad spectrum of applications like individual method qualification and validation studies or stability studies.
VelaLabs is not limited to the below-mentioned applications, contact us for your specific needs!
End-Point PCR:
- Presence/absence testing
- Genetic modification confirmation
qPCR:
- Residual DNA/RNA testing (E. coli, HEK293, CHO,…)
- Gene expression analysis
- Vector copy number determination
- Detection of microbial contamination
Principle of PCR:
By PCR (polymerase chain reaction) a DNA or cDNA template is amplified more than thousandfold. This is achieved by an initial denaturation step at 94 °C or higher to separate the intertwined DNA strands, followed by primer annealing at 40 °C – 60 °C. Primers are short synthetic DNA fragments that offer the target region for the DNA polymerase. In the final extension step, the temperature is raised again, and the DNA polymerase extends the primers, synthesizing the new strand. Denaturation, annealing, and elongation constitute a single PCR cycle where the DNA is doubled. The reagents for a successful reaction come along in a master mix containing a thermostable DNA polymerase, MgCl2, dNTPs and a buffer. Gel electrophoresis as endpoint analysis serves to detect and quantify the amplified sequence of interest.
Principle of qPCR:
Robust target quantification in real-time is performed by RT-qPCR (quantitative reverse transcription polymerase chain reaction). Fluorescent dyes added to the master mix, that hybridize with the amplified product, enable a specific measurement of the target sequence. Real-time PCR systems detect the fluorescence intensity above the background signal (Cq value) at the end of each PCR cycle to quantitate the newly synthesized DNA strands via a standard curve (dilution series of a template with known concentration). Quantitative PCR has applications across various fields including gene expression analysis and absolute quantification
Instruments at VelaLabs:
Thermocyclers:
With our state-of-the-art instruments we offer precise quantification for various individually adapted assays in a 96-well format:
- QuantStudio 5 Real Time PCR system (Thermo Fisher Scientific)
- CFX96 Touch Real-Time PCR Detection System (Bio-Rad)
Additionally, we have the following specialized equipment:
- Azure 300 Gel Imaging System (Azure Biosystems):
- Precise visualization of DNA bands using UV and blue-light detection, ensuring sharp band resolution, accurate documentation, and compliance-ready image export.
- NanoDrop 2000 Spectrophotometer (Thermo Scientific):
- Nucleic acid quantification and purity assessment as sample preparation for downstream applications
- Qubit Fluorometer (Thermo Scientific):
- RNA integrity assessment for reliable evaluation of RNA quality prior to downstream applications ensuring data robustness and reproducibility
FAQs about PCR/qPCR
Why is qPCR important in analytical testing?
qPCR offers high sensitivity, specificity and fast turnaround times for nucleic acid analysis. It is widely used for viral load determination, gene expression analysis, contamination control and detection of microorganisms.
What is PCR used for?
PCR (polymerase chain reaction) is used to amplify specific DNA sequences, allowing detection of very small amounts of genetic material. It is commonly applied in microbiology, molecular biology, viral testing and identity analysis.
What is the difference between PCR and qPCR?
Conventional PCR detects amplified DNA at the end of the reaction, while qPCR (quantitative PCR) measures amplification in real time during each cycle. qPCR therefore allows both detection and quantification of nucleic acids.