Characterization of sub-visible protein aggregates in biotherapeutic formulations.
Dynamic Light Scattering, Molecular size, Particle concentration, Particle size, Protein Aggregation, Tech Talk, Zetasizer »
How can I use DLS for sample xyz?
What sample concentration / refractive index is needed for reliable DLS size measurements?
Has it been done before?
In Dynamic Light Scattering (DLS) intensity fluctuations in the sample are analyzed to obtain size information on the sample. The technique is applicable to a wide size range from sub-nanometer up to a few microns. In order to obtain a meaningful result, a minimum scattering signal strength must be present. Malvern’s Zetasizer Nano software contains a calculator to help estimate the required minimum concentration.
If anyone else has …
α-synuclein promotes vesicle assembly – when size can support theory
What is a vesicle?
In cell biology, a vesicle is an (often spherical) assembly of amphiphilic molecules (usually proteins and phospholipids), and is typically sized between 30 nm – 100 nm in diameter. Vesicles are often classified by the type of interaction they are involved with. For extracellular vesicles (or exosomes), this is usually intercellular signal transmission. A particular example of exosomes is synaptic vesicles, which transmit signals between neurons (average size: 40 nm).
In a paper entitled “Structural basis of synaptic vesicle assembly promoted …
Dynamic Light Scattering, Electrophoretic light scattering, Meet the Experts, Particle size, Protein Aggregation, Zeta potential, Zetasizer »
Adjuvants for vaccination – How size and zeta may be relevant
What is an adjuvant?
Adjuvants are substances which are added to medicines to trigger and enhance an immune response to an antigen. In the USA, the most commonly-used adjuvant is alum, and many vaccines in use today contain some aluminum components to help the vaccine work better. While the exact mechanisms of how adjuvants work is unclear to some extent, a typical adjuvant component is much larger than the antigen or antibody itself, often around 100 nm in radius.
In a paper entitled …
Dynamic Light Scattering, Nanoparticle Tracking Analysis, Nanosight, Particle concentration, Particle size, Protein Aggregation, Tech Talk, Zetasizer »
Nanoparticle Tracking Analysis or Dynamic Light Scattering ?
After a recent joint NanoSight and Zetasizer demonstration, I received this set of questions that helps illustrate the core of the issue of these two techniques:
Since the Nanosight gives me particle concentration, it’s somewhat straightforward to calculate what % of my Nanoparticles are aggregated. Can I calculate % aggregated particles in the DLS as well? Or does the varying amount of intensity, and the protein+nanoparticle mixture in the sample confound that calculation?
If I wanted to measure non-spherical particles (for example nanorods), which system …Read the full story »
Nanoparticle Tracking Analysis, Nanosight, Particle concentration, Particle size, Protein Aggregation, Tech Talk »
We recently published our application note, “Adeno-Associated Virus titer and aggregation characterization“, which discusses a new NanoSight method that allows the visualization and therefore analysis of viruses smaller than what was previously possible to measure. This represents an exciting extension of the Nanoparticle Tracking Analysis (NTA) technique in response to market demand for tools for better characterization of small virus types.
Adeno-Associated Virus (AAV) is commonly used in the gene therapy field as a delivery vector. Tools to characterize both the concentration and purity or aggregation of the samples are limited because the primary virus size is …
Differential Scanning Calorimetry, Electrophoretic light scattering, Isothermal Titration Calorimetry, Label-free analysis, Malvern Events, MicroCal, Microcalorimetry, Molecular structure, Protein Aggregation, Raman spectroscopy, Zetasizer »
I had the pleasure of participating in the 5th International Symposium on Higher Order Structure of Protein Therapeutics, also known as HOS2016, sponsored by CASSS. The meeting was at the Renaissance Hotel in Long Beach, California April 11-13, 2016. I was joined by my Malvern colleagues Rajib Ahmed and David Sanborn.
The HOS Symposium is a platform for open discussion of development and optimization of analytical techniques to study HOS of proteins and biopharmaceutical products (including monoclonal antibodies and antibody-drug conjugates), biocomparability, and biosimilars.
Protein higher order structure (HOS) includes the secondary, …
Dynamic Light Scattering, Molecular size, Molecular weight, Protein Aggregation, Tech Talk, Zetasizer »
How do I know the minimum concentration needed for measuring the size of my protein?
We are asked this question all the time when we are demonstrating the Zetasizer. Happily, if the Zetasizer software is installed (and it can be downloaded for free, by the way) there is a minimum concentration calculator integrated – so it’s simple! The calculator can be accessed from Tools – Calculators – Concentration Utilities – Minimum Concentration Calculator
Let’s look at a few examples to get an idea of some typical values for your samples. The left …
How can a quartz cuvette be properly cleaned?
The Zetasizer has a flow cell [ZEN0023] used in conjunction with either the titrator, the NanoSampler, or as a chromatography or field flow fractionation detector. A similar flow cell [ZMV1008] is also in use with the Zetasizer microV when used as a molecular weight detector in conjunction with the OmniSEC software. Advanced cuvettes may also be used in UV-Vis, fluorescence & circular dichroism spectrophotometers, MALLS as well as goniometer systems and other specialized optical detection. If the signal from the quartz cell is …
Often, pharmaceutical formulations contain therapeutic proteins at relatively high concentration. Under such conditions, the stability of the protein as well as the formulation overall is of significant concern. Great effort is invested in optimizing stability, and particular attention goes to the choice of buffer components. Various components might form part of the ‘final’ recipe including cosolvents (such as polyethylene glycol (PEG), glycerol, ethanol) to reduce the dielectric constant of the buffer and influence electrostatic interactions, chelating agents (citric acid or citrate salt buffers, ethylene-diamine-tetra-acetic acid (EDTA), tris(hydroxymethyl)aminomethane (TRIS), tartaric acid) and …Read the full story »
We recently published a new application note on protein characterization using our latest SEC system, OMNISEC, and I wanted to bring your attention to this elegant piece of work that beautifully shows the kind of information you can get from a multi-detector system and, perhaps more importantly, the kind of information you might be missing from a single-detector, UV-based system.The chromatogram below shows a typical chromatogram of a standard protein, β-amylase:
It looks pretty typical with a monomer and a small amount of aggregates, right? Wrong!
Let’s looks again with a multi-detector …
Can one calculate a PDI value excluding a small aggregation peak? For example if the main particle species is ~100nm and there is a small contribution (2% by intensity) from a peak at 5 microns, is it possible to recalculate the z-average and the PDI ignoring the 5 micron peak. Alternatively, is it possible to determine a PDI for the smaller species only?
The z-average will be weighted more towards smaller components, because only the initial part of the correlation function is fit. Following the ISO method to determine the z-average …
Dynamic Light Scattering, Meet the Experts, Nanoparticle Tracking Analysis, Protein Aggregation, Raman spectroscopy, Resonant mass measurement, Size Exclusion Chromatography, Tech Talk »
I recently took part in a question and answer session with BioPharm International about techniques for measuring and characterizing protein aggregates. The full discussion can be viewed here, however I thought I would also use this blog post to summarize the main points.
Essentially, there is a pressing need within the biopharmaceutical industry to understand aggregation pathways, and the risk factors that can induce aggregation, right from the start of the drug development process to align manufacturing processes with Quality by Design (QbD) principles. Given the link between protein aggregation and …
Light scattering in chromatography can sometimes be challenging: The scattering signal is proportional to the size to the 6th power, and that is why particles in the mobile phase can lead to noise in the light scattering detector. Now we have special light scattering columns available specifically designed for low shedding, making them particularly well suited for analytical chromatography of proteins and peptides. The Viscotek PLS-columns are silica-based columns for the separation of proteins for light scattering applications. Their excellent stability minimizes particle shedding to maintain light scattering baselines as …Read the full story »
There is one, and it is called the NanoSampler.
The Zetasizer has been around for a few years, and the Zetasizer series now includes several models (90 degrees, backscattering, size and zeta, flowmode for chromatography detection, automated plate sampler). A very recent accessory which is compatible with any Zetasizer Nano in the field is the autosampler or with its full name the “Zetasizer NanoSampler“.
In dynamic light scattering (DLS) the size distribution of molecules or particles can be obtained in a relatively short time. The technique is therefore ideal for automation, because preparing …
Last week we offered a webex presentation titled “Speed your way through protein formulation screening by automating your measurements: A2/B22 with the Zetasizer APS”
The presentation had a focus on protein formulation, where often the objective is to assess protein stability as a function of various parameters. Here, light scattering can be used as a tool to assist in screening conditions:
Monitor: Aggregation temperature, Dynamic Light Scattering (DLS) vs temperature
Predict: Interaction parameter, DLS vs concentration
Predict: Second Virial Coefficient, Static Light Scattering (SLS) vs concentration
While the Zetasizer APS has previously been used for dynamic …Read the full story »
Protein Aggregation, Protein Mobility, Tech Talk »
Our portfolio of instruments to aid in biopharmaceutical research has grown significantly in recent years, thanks to innovation from our Bioscience Development Initiative as well as acquisitions of NanoSight, Viscotek and most recently, MicroCal. As a result,the amount of useful resources we have created for those interested in analyzing and characterizing proteins has also exploded.
This list guides you through 20 essential resources for the protein scientist at all stages of research & formulation development. I hope you find it useful. To explore our full, free archive of articles, whitepapers, application notes …
Meet the Experts, Protein Aggregation »
Hello – I’m Stéphane Rouquette; I’ve worked for Malvern instruments since 1991, and my first role within the company was to sell airborne and liquid particle counters for cleanroom testing and QC for injectables (USP788). I then continued my journey with Malvern by selling Mastersizer & Zetasizer instruments, before moving into the role of sales manager for France. I eventually became the country manager, and two years ago, I decided to focus on the Biosciences arena. I now lead European sales for Malvern Instruments in this quickly growing market.
In April. I’m going to be travelling to …
Malvern Events, Nanosight, Protein Aggregation, Viscotek, Zetasizer »
Greetings, Materials Talks readers, from sunny Palm Springs in beautiful Southern California! I wanted to update you on the wonderful conference Malvern has attended here: “Peptalk – The protein science week.”
Peptalk, held each January, is dedicated to the fields of protein and peptide therapeutic development. This year’s event attracted over 1,200 attendees from all over the world. Presentations were divided into seven pipelines covering everything from protein and antibody development, formulation stability, purification and aggregation, all the way to facilities design, manufacturing and packaging.
Malvern was one of 80 suppliers vying …