Expert Protein Analysis Services
Protein Characterization

Protein Characterization

Proteins are complex molecular entities derived from biological processes. They differ from each other in their size, molecular structure and physiochemical properties. These differences allow for protein analysis and characterization by separation and identification.

In th product processs they are almost always not fully purified and homogeneous molecules, but instead show a certain degree of structural heterogeneity. This means that the characterization of a protein always has three aspects:

•    Identifying the major protein component
•    Identifying the minor components
•    Quantifying the minor components (both process- and product-related impurities)

Separation is typically done via electrophoresis where proteins are differentiated by size or mass, and isoelectric focusing, where protein are separated by charge. These techniques can be done independently or in combination-referred to as 2D electrophoresis. Equipment for separation includes polyacrylamide gels, organic stains, electrophoresis boxes, isoelectric focusing immobilized strips, 2D electrophoresis equipment, and protein standards. Identification is done via mass spectrometry where molecules are ionized to determine their mass to charge ratios. Mass spectrometry equipment includes protein fractionators, GC/MS, LC/MS, CE/MS and time of flight systems. Identification can also be done via amino acid sequencing by Edman degradation, crystal imaging and surface plasmon resonance. With so many options available, laboratories typically will employ multiple strategies for protein analysis and characterization.

Protein Quantification

Accurate protein quantitation is is necessary to understand the total protein content in a sample or in a formulated product and is essential to protein studies in a variety of research topics. A wide array of different methods have been developed to quantify complex mixtures of proteins as well as a single type of protein. How much protein is existing? It may seem an easy question, but in practice protein quantification is a difficult task. Being a sensitive measurement, large errors are easy to make and there are only a limited number of accurate methods available.

We’ve put a lot of work into developing and optimising the methods we use. Now, we offer our own specially developed techniques that give high precision measurements and accuracy.
Furthermore, we cross-validate our methods so that you don’t have to just take our word for it. We can show that our methods do what we said they would.
So if you need to know the precise quantity of a protein in your drug development or research project, you can choose from the following services.

  • Amino Acid Analysis (AAA), to determine the amino acid composition of a protein without using an external standard.
  • Extinction Coefficient (A280), to calculate protein concentration by measuring UV absorbance at A280. This technique helps demonstrate consistency and comparability between batches.

Note that protein concentration can also be determined by ELISA assays or mass spectrometry using absolute quantification (AQUA).

Protein quantification is a key part of ICH Q6B.

  • Amino Acid Analysis (AAA)
  • Extinction Coefficient (A280)

Amino Acid Analysis (AAA)

Calculate content with confidence - Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. Sometime during the mission to develop a biopharmaceutical drug or other research application, it is nessesary to find out exact protein quantities. The reference method to quantify peptides and proteins is amino acid analysis (AAA). Protein content must be measured as accurately as possible because it is such a fundamental parameter that will influence all other quantification results obtained with other methods. Even a slight deviation can lead to incorrect results and, ultimately, to an unsuitable product.

This is where Protagen Protein Services (PPS) can support our customer. We are comitted to quality, precision and accuracy. We always back up our work through cross-validation.

Standard amino acid analysis (AAA), including hydrolysis, is a wonderful tool for determining the precise protein content or concentration of a sample without the use of an external standard.
But AAA is an even more powerful tool when carried out with our time-resolved hydrolysis method, which will give the precise amino acid composition of a protein. We typically use this for the characterization of a reference standard.
Amino acid analysis is a key part of ICH Q6B and we carry it out according to PharmEu.

Technical information

  • Amino acid analysis (AAA) can be applied in many ways to reveal different aspects of your protein. Here are some examples. In combination with UV-absorbance measurements, it can be used to directly determine the extinction coefficient of a protein.
  • If we skip hydrolysis, it can be used to quantify free amino acids, for example in cell culture media.
  • It can quantify unusual amino acids such as Norleucine (encountered in E. coli fermentations) or Hydroxyproline and –lysine.
  • To determine concentration based on mass, it can be combined with MALDI-TOF to reveal the intact mass of the protein.

Protein Modification

One of the important areas of protein characterization is to identify those proteins that are post-translationally modified and their modification sites, and to characterize the function of the modifications. Almost all proteins, whether produced and purified as recombinant proteins or isolated from natural sources, will carry to some degree modified amino acids.

Characterizing product-related impurities is a major task. You’ll need us to analyze them with as much sensitivity and accuracy as possible. Such modifications may of course be part of the physiological function that you are developing.

But we understand that you’re keen to observe the lowest possible number of process-related modifications. The latter may occur during the sample handling process, for example in proteins that have been stored for a period of time during a stability study. They are unintentional and a source of structural heterogeneity in a protein. And that’s not what you want when you’re trying to develop a consistent, trustworthy drug substance or product.

All such changes are known as post-translational modifications and whilst some of them are desired, others are unwanted.

At Protagen, we use our mass spectrometers  to detect even the smallest and peskiest modifications, which we usually analyze using a combination of electrophoretic, HPLC and peptide mapping methods.

Normally, mass spectrometry data is qualitative rather than quantitative. However we’ll provide you with data evaluations – that is, real numbers about the degree of modification taking place in your protein – so that you get a clear picture of how your protein is doing.

You can choose from the following types of analysis:

  • Deamidation
  • Oxidation
  • Glycosylation
  • Phosphorylation
  • N- and C- terminal truncation (see N/C- Terminal Sequencing)
  • Acetylation
  • Pyroglutamate formation


Characterizing post-translational protein modifications is a key requirement of ICH Q6B.

  • Deamidation
  • Oxidation
  • Other Modifications

Deamidation

The trouble with small changes - The trouble with tiny modifications in a complex molecule is the difficulty in trying to find them.
This is certainly the challenge when proteins – particularly those containing the amino acids asparagine or glutamine – have undergone deamidation. It results in the addition or removal of as little as one Dalton of molecular weight to the entire protein structure and it’s very difficult to pick up. Then there’s the issue of attempting to work out if the modification is really due to deamidation or simply due to variations in isotopic distribution, which is normal and occurs naturally in peptides. Our high-resolution mass spectrometers, coupled with an eye for detail and experienced bioinformatics team, enable us to distinguish between the two causes with reliability and high accuracy.

Furthermore, we can determine the specific sites of deamidation by undertaking peptide mapping using a high-resolution mass spectrometer and mass accuracy such as LC-ESI-MS/MS.
Analyzing deamidation is a key requirement of ICH Q6B.
 

Technical information

Deamidation is commonly observed as a post-translational modification of the amino acid asparagine, which turns into a mixture of isoasparate and aspartate via a succinimide intermediate. The rate of modification is influenced by factors such as the presence and interaction of surrounding amino acids, pH and temperature buffers. Deamidation of glutamine also occurs, but much less frequently.
You will be interested in finding out whether your protein has undergone deamidation because it leads to a change in isoelectic point (pI) and therefore the presence of charge heterogeneity. We can reliably observe pI by 1D-IEF or IEX-HPLC.

Glycosylation

Do you need to investigate glycosylation to ensure protein structural integrity during drug development? Would you like to see if any molecular alterations have taken place during fermentation, purification or storage?

Proteins that have undergone glycosylation are incredibly complex. Several glycans may be attached to numerous amino acids, and each glycan will show unique and sometimes unexpected branching.

You’ll want only the most experienced researchers looking at this in your proteins because of the intricacy involved. This is where our long history in specialised biopharmaceutical protein analysis is of benefit to you.

Although glycosylation analysis is a fairly new field of research, we have such an in-depth understanding of protein structure that we’re already unparalleled in terms of the level of detail, such as rigorous structural information, we can provide.

We apply a selection of techniques, depending on your circumstances, in order to obtain a complete overview of the glycans in your proteins. They include high resolution mass spectrometry, HPLC and electrophoretic methods.

Mass spectrometry is an important technique because of its high selectivity and sensitivity. It also provides information quickly. The mass spectrometry strategy we’ll employ will depend on the level of structural information you require. A typical strategy is shown below.

 

Once the analyses are complete, we package the results to tell you exact glycosylation sites and how the glycans are or are not contributing to the product you’re developing.

You can choose from the following types of analysis:

  •     N-linked glycosylation, to explore the attachment of N-linked glycans at the end of enzymes.
  •     O-linked glycosylation, to investigate the more complex addition of O-linked glycans, which occurs later in protein processing.
  •     Sialic acid content, to determine the type and quantity of sialic acid present at any stage of drug development, information which is helpful in further glycosylation analysis.
  •     Monosaccharide content, to determine the composition and quantity of N- and O-linked glycosylation.
  •     Glycosylation site, to work out exactly where glycans are attached to your proteins, because normally not all available sites will be occupied.

Characterizing glycosylation is a key requirement of ICH Q6B.

Technical information

Protein glycosylation is a post-translational modification that alters a protein’s structure or function. It is a very common and biologically relevant modification that may be directly involved in the formation of diseases. It also plays an important role in altering the pharmacokinetics by stabilizing the protein conformation, improving solubility or protecting from proteases.

The main challenge in the analysis of protein glycosylation is its structural complexity, which arises from a combination of factors such as:

  •     Multiple glycosylation sites per protein
  •     Multiple glycan structures attached to each site
  •     The complex, branched structure of glycans
  • N-linked Glycosylation
  • O-linked Glycosylation
  • Sialic Acid Content
  • Monosaccharide Analysis
  • Glycosylation Site

N-linked Glycosylation

Together with our clients, we’re expanding in the biopharmaceutical field to build a portfolio of methodologies for glycan analysis. Our aim is to provide the best combination of methods to suit your particular purpose.

When compared with O-linked glycosylation, the analysis of N-linked glycosylation is fairly straightforward, as scientists have been researching it for a longer period of time. The fastest, most accurate and most economical way is through the use of our mass spectrometers.

Questions you might have about glycosylation that can be answered by looking at N-linked glycans include “Is my protein glycosylated?”, “What do the glycans look like?” and “Are they helping or hindering the development of my drug?”

For example, you may like us to determine the structure and heterogeneity of glycosylation for an entire protein or for each potential glycosylation site. To determine the structure of N-glycans we’ll use mass spectrometry (MALDI or LC-ESI) after labelling or permethylation. But to determine the antennary of glycans, or to quantify glycans, we’ll use HPAEC-PAD. Being a typical HPLC method, it has the advantage that you then can easily validate the assay for release testing. The Normal-Phase HPLC (NP-HPLC) complements this setup by providing an additional, highly sensitive and high-resolution analysis of your samples.

You might also ask us to find the sites of glycan attachment. We will do this either by peptide mapping or MALDI-ISD.

Other information about a protein’s glycosylation can be combined with what we know about its N-linked glycans. For example, we can determine the composition and amount of glycans attached to a protein by monosaccharide analysis and sialic acid analysis. The structural heterogeneity of a glycoprotein can be visualized using 2D PAGE or intact mass.

The characterization of protein glycosylation is a requirement of ICH Q6B.

Technical information

N-linked glycans are a common feature of glycoproteins such as EPO, antibodies and FSH. They have been shown to be important in many ways. They have an influence on protein folding as well as other factors, such as protein solubility, stability, immune reactions and cell-to-cell interactions.

N-glycosylation increases the structural heterogeneity of proteins when a range of structurally similar - but not identical - glycans are attached to each glycosylation site.

Glycosylation may be directly involved in the formation of diseases, but also plays an important role in altering the pharmacokinetics by stabilizing protein conformation, improving solubility or protecting from proteases.

The N-glycosylation profile of a recombinant protein can vary largely depending on the organism chosen for expression.

Glycosylation may be directly involved in the formation of diseases, but also plays an important role in altering the pharmacokinetics by stabilizing protein conformation, improving solubility or protecting from proteases.

The N-glycosylation profile of a recombinant protein can vary largely depending on the organism chosen for expression.

Protein Primary Structure

A protein is more than the sum of its amino acids - When looking at a protein in drug development, the first step you’ll most likely want to take is an analysis of its amino acids and covalent bonds, or primary structure.

Our scientists meticulously characterize primary structure by using several separate but interlinked analysis techniques. They cross-validate the findings so that you don’t just receive the stand-alone results of each of the techniques, but an integrated report showing how the results combine to tell you more. This is something that we at Protagen Protein Services (PPS) do exceptionally well.

We are well versed in the most important techniques used to analyze primary structure today. Your project may benefit from a couple or more; we’ll work with you to design a methodology that best suits your needs.

  • Peptide Mapping
  • De-novo sequencing
  • Intact mass
  • N/C terminal sequencing
  • Disulfide linkage

Not just a protein identity test - Peptide mapping is both a regulatory requirement under ICH Q6B and a powerful tool for understanding how a protein may be modified to suit your purpose.

It provides a whole suite of information to help inform the drug development process.

One of the key benefits of engaging us in your research projects is that we’ll both fulfill regulatory requirements and provide you with full data interpretation, giving you all the information we uncover.

We pioneered the mass spectrometry analysis service for the biopharmaceutical industry and have been leaders for almost 15 years, making us your peptide mapping specialists.

Our bioinformatics staff, who dedicate their time to analyzing mass spectrometry outputs, know that there are a number of ways in which peptides can be mapped in a laboratory. But each method has strengths and weaknesses depending on your situation.

We design the study so that we are using the method with the greatest strengths for your specific project. Other protein service providers don’t have a similar breadth of options when it comes to peptide mapping with mass spectrometry.

Mapping peptides was once the domain of chromatography and UV research. However, these simple methods of analysis did not allow for the identification of individual peptides without great difficulty.

Now, with our superior understanding of modern mass spectrometry, we can identify and analyze individual peptides within a protein with great certainty. And because Protagen’s scientists were at the forefront of mass spectrometry software development when it emerged almost 15 years ago, clients benefit from our finely tuned skills in this area.

Technical information

Peptide mapping is commonly undertaken to analyze the protein primary structure after cleavage of the protein into proteolytic peptides. The power of peptide mapping lies in the large number of site specific molecular features that can be detected.

When using one digestion enzyme (for example, Trypsin), peptide mapping is typically carried out for protein identification. The analysis is performed using MALDI-MS/MS or LC-ESI-MS/MS, for example during protein identification after electrophoresis, such as 1D or 2D PAGE.

When using multiple enzymes, peptide mapping is applied for the confirmation of a complete amino acid sequence, for example when confirming the amino acid sequence of a biosimilar and comparing it with the originator molecule.

Depending on the experimental setup, peptide mapping can also be used to determine the N- and C-terminus of a protein. This is sometimes crucial information for our clients, for example in the case of monoclonal antibodies, where the truncation level of the C-terminal Lysine can be monitored.

Proteins and peptides are ionized and fragmented in the mass spectrometer. The resulting MS/MS spectra is used to sequence the individual amino acids in order of their appearance in a protein or peptide.

In combination with additional sample preparation, peptide mapping can also be used to determine:


If the protein sequence is not found in a protein database, it can be deduced by de-novo sequencing.

Up to 100% sequence coverage confirms the amino acid sequence. The identification is based on LC-ESI-MS/MS data, which allows the sequencing of peptides with high confidence. Overlapping peptide sequences confirm the order of amino acids in the sequence.

Impurity Charaterization

Despite purification respectively polishing processes in protein preparation, it’s not possible to create proteins without host cell proteins. In addition to these process-related impurities there are also product-related impurities to be caused by protein modifications. 
Protein impurities may generate undesired side effects. So characterizing protein impurities is a key requirement of ICH Q6B.

We identify and quantify all the proteins in your mixture using mass spectrometry. By carrying out a bioassay, we can also see if the impurities are influencing the efficacy. Finally, we eliminate the unwanted proteins one step at a time by focusing on the differences in their characteristics. The process is tailored to your needs because every client’s requirements are different.

 

  • Product-related impurities
  • Process-related impurities
  • Protease activity using customized FRET technology

Characterizing protein impurities is a key requirement of ICH Q6B.

We can search for: Product-related impurities, which are basically types of protein modification. Examples are deamidation or oxidation. We analyze these with a combination of 2D gel electrophoresis, high performance liquid chromatography (HPLC)and peptide mapping.

Applications in Pharma-Biotech

  • Optimization and characterization of Up-Stream and Down-Stream-Processing (USP&DSP) steps in the context of protease burden
  • Characterization of protein-containing or protein-based drugs
  • Comparability of protein-containing or protein-based drugs
  • Assessment of presence of proteases in drug product causing stability issues

 Application in Diagnostics

  • Quality control of protein-based raw materials such as Serum Albumin or IgG molecules
  • Quality control of complex buffers or of final immuno assay used as diagnostic kit

 

Publication:
Development of a fluorescence resonance energy transfer peptide library technology for detection of protease contaminants in protein-based raw materials used in diagnostic assays; Kapprell HP, Maurer A, Kramer F, Heinrich B, Buenning C, Narvaez A, Kalbacher H, Flad T (download

Higher Order Structure

Developing the formulation of a biopharmaceutical can be challenging and a solid understanding of the structure and conformation of your product is essential. Correct higher order structure (HOS) is critical to ensuring proper functionality, activity, and stability of a biopharmaceutical product. A well-developed panel of methods for HOS characterization is an essential component of a complete product characterization program.

Different formulations may alter the secondary and tertiary structure of a biopharmaceutical, which in turn can affect protein activity. PPS provides analytical methods to analyze the structure and stability of your biopharmaceutical in its formulation.

  • Analytical ultracentrifugation (AUC)
  • Differential Scanning Calorimetry (DSC)
  • Circular Dichroism (CD)

Analytical ultracentrifugation (AUC) separates protein species directly in solution, without the use of a stationary phase such as in size-exclusion chromatography (SEC). The sedimentation rate of the molecule(s) is induced by the centrifugal force, and is monitored continuously by UV absorbance, fluorescence, or interferometry to produce a size distribution profile of the species present within the test sample. Sedimentation velocity analytical ultracentrifugation (SV-AUC) generates sedimentation coefficient values and reports the relative percentages of monomer, multimer, and aggregate species. Information on molecular weight (MW) and hydrodynamic shape are also obtained.

Fluoresence

Fluorescence utilizes the natural intrinsic fluorescence of Tyrosine (Tyr) and Tryptophan (Trp) residues and provides information on the local environments around these residues. Fluorescence is an excellent comparative tool and is complementary to CD and FT-IR.

The below image shows the relative intrinsic fluorescence of Trp and Tyr residues for a mAb product in its formulation buffer (Left: 280nm excitation. Right: 295nm excitation)

 

Protein Nuclear Magnetic Resonance (NMR)

BioPharmaSpec recommends NMR analysis of biopharmaceutical products for the high-end assessment of higher order structure (HOS). The US FDA has also stated in numerous presentations that NMR is used in their laboratories to provide a detailed assessment of HOS. NMR is an orthogonal assessment, alongside CD analysis, for secondary and tertiary structure determination. The technique is particularly useful in the early stages of biopharmaceutical development and for initial comparability assessments.

BioPharmaSpec’s NMR service comprises 1D 1H-NMR for an initial assessment (and for analysis of products at too low a concentration for 2D-NMR) followed by 2D-NMR (normally 1H – 13C 2D NMR analysis). BioPharmaSpec recommends 1H – 13C 2D NMR analysis over 1H – 15N 2D NMR analysis because of the higher natural abundance of the 13C isotope which provides a more significant response, particularly for products larger than 50kDa.

During NMR analysis, samples are supplemented with D20 and 1D 1H spectra followed by 2D 1H-13C heteronuclear single quantum coherence (HSQC). NMR spectra are recorded at approximately 298K on a Bruker DRX800 equipped with a triple resonance cryoprobe.

 

Fourier Transform InfraRed Spectroscopy (FT-IR)

InfraRed (IR) spectra provide qualitative and quantitative information on the secondary structure of proteins such as α helices, β sheets, β turns and disordered structures.
The most informative IR bands for protein analysis are amide I (1620-1700 cm-1), amide II (1520-1580 cm-1) and amide III (1220-1350 cm-1).
Amide I is the most intense absorption band in proteins and consists of stretching vibration of the C=O (70-85% and C-N groups (10-20%).

Amide II is governed by in-plane N-H bending (40-60%), C-N (18-40%) and C-C (10%) stretching vibrations.
FT-IR provides an orthogonal assessment of secondary structure to Far UV CD analysis. It is often considered more useful than CD for products with high levels of α helices and β sheets because, unlike Far UV CD, FT-IR does not show a disproportionately high response to α helix.
The profiles obtained and the fitting data can be used to assess the comparability of the secondary structure of different batches or formulations, and between originator and biosimilar samples.

Overlay of FT-IR spectra at 2200-1000cm-1

SERVICES

Protagen Protein Services (PPS) is a world leading CRO and recognized expert for analytical services in protein science regarding characterization, method development, validation and routine testing of antibodies, proteins, vaccines and other formates of biopharmaceuticals. Our sites are equipped with modern, state-of-the-art equipment for the analytical characterization of biopharmaceuticals regarding structure, purity, chemical modification, aggregation, particle formation, thermal stability e.g.. We offer service modules for analytcal support from research to approval regarding:

  • Developability
  • Clone Selection & Process Development
  • Extendend Characterization & Comparability
  • Stability & Release

READ MORE


Protagen Protein Services (PPS) is a world leading CRO and recognized expert for analytical services in protein science. More than 20 years of market experience and the comprehensive spectrum of validated analytical methods ensure the highest quality for customers in the pharmaceutical, biotech and life science industry.

PPS supports Biosimilar developers with a broad range of analytical methods and consulting in achieving and demonstrating Biosimilarity