Assessing and monitoring the quality of therapeutic mAb products
Question: Which analyses should be part of mAb characterization? I need to ensure the quality and consistency of my mAbs. Which analyses should I conduct for proper mAb characterization? Answer: For several years, monoclonal antibodies (mAbs) have been the fastest-growing type of pharmaceutical molecules [1]: More than half of all new drugs approved from 2015 […]
Latest in: Analysis of Host Cell Protein See all
Critical reasons why mAbs call for orthogonal HCP analysis techniques
Question: HCP-ELISAs show our mAb is pure. So why do we need orthogonal analysis? We produce a monoclonal antibody (mAb) product,
Investigation of ubiquitin in purified antibody products
Question: Should we worry about ubiquitin in our mAb? Our process-specific HCP ELISA did not detect any Host Cell Proteins (HCPs) in our purified monoclonal antibody (mAb). However, mass spectrometry-based HCP analysis identified ubiquitin in low-ppm amounts. Do you have any suggestions for why the Protein A purification did not remove it? Why did ELISA not detect it? And should we worry about
Why measure process-related impurities in gene therapies with LC-MS?
Question: Can I use mass spectrometry to document process-related impurities? I work in a biopharmaceutical company developing a gene therapy product
Latest in: Analysis of Biopharmaceuticals See all
Speedy and detailed protein ID of SDS gel bands
Question: Can I identify hundreds of gel bands in a few days? The biopharmaceutical company that I work for, consistently uses SDS PAGE and Western Blotting to check protein expression. But this means we spend a great deal of money on antibodies, which I feel may be unnecessary. In addition, there are several unknown proteins in the gels, possibly degradation products, which
Assessing and monitoring the quality of therapeutic mAb products
Question: Which analyses should be part of mAb characterization? I need to ensure the quality and consistency of my mAbs. Which analyses
How to characterize antibody glycosylation by HILIC-MS
Question: How do I characterize the N-glycosylation of my antibody? What is N-glycosylation and how do I characterize the N-glycosylation of my antibody? Is it possible to compare batch-to-batch variations of antibody glycosylation? Answer: Glycosylation is a post-translation modification (PTM). It occurs when a carbohydrate moiety (glycan) is added to the protein. Since there are different types of glycosylations, you distinguish them by the glycan
Latest in: N- and C-Terminal Protein Sequencing See all
Why is my protein’s N-terminal blocked (and what can I do about it)?
Question: Does a blocked N-terminal hinder Edman degradation? I found that the N-terminal of my protein is blocked. So, how can I analyze it by Edman degradation / n-terminal sequencing - and what causes the problems? Answer: To sequence the protein N-terminal is a requirement according to the ICH Q6B Guideline. This includes structural characterization of recombinant proteins for clinical testing, and demonstration of
3 Techniques to find the protein N- and C-terminal sequence
Question: How do I sequence the protein n-terminal and c-terminal? Where does the amino acid sequence start and where does it end?
Preparing samples for N- and C-terminal sequencing by MALDI ISD
Question: How do I prepare samples for MALDI ISD analysis? I am interested in your N and C-terminal sequencing using MALDI ISD. How do I prepare the sample for analysis? Answer: MALDI In-Source Decay (ISD) is used for confirmation of the N- and C-terminal of known proteins, therefore you would need to supply us with the sequence of the protein you are working with
Latest in: Analysis of Amino Acid in protein See all
Why you need accurate concentration determination of protein standards
Question: How do I quantify my protein standard? How do I quantify my protein standard? Is it true that it if it is done incorrectly it can affect the determination of my protein concentration? Answer: Have you ever questioned the quantitative data you get from Bradford, Lowry or BCA protein quantification assays? These traditional methods are very fast and can be performed in most
Determine the Molar Extinction Coefficient of protein in 3 small steps
Question: How do I determine the molar extinction coefficient of my protein? I have a purified protein that I would like to
4 things to remember about Amino Acid Analysis of proteins & peptides
Why perform Amino Acid Analysis? Amino acid analysis of proteins is a method to determine the absolute amounts of individual amino
Latest in: 1D & 2D Electrophoresis See all
Speedy and detailed protein ID of SDS gel bands
Question: Can I identify hundreds of gel bands in a few days? The biopharmaceutical company that I work for, consistently uses SDS PAGE and Western Blotting to check protein expression. But this means we spend a great deal of money on antibodies, which I feel may be unnecessary. In addition, there are several unknown proteins in the gels, possibly degradation products, which
1D SDS PAGE versus 2D PAGE. Any recommendations?
Question: Should I use 1D or 2D SDS PAGE? I’m interested in isolating and identifying cell surface receptor proteins. They typically have high
How much protein should I load for protein identification by mass spec
Question: How should I prepare my samples for 2D electrophoresis? How should I prepare my samples for 2D electrophoresis, and how much protein should I load to get protein ID by mass spec? Answer: Protein samples for 2D PAGE should not contain high salt concentrations, ionic detergents like SDS, or cellular debris and DNA from cell lysis, as these will disturb isoelectric focussing (IEF)
Latest in: Bioinformatics See all
GPMAW lite – Protein Sequence Calculator (and much more)
When working with proteins you often need to make simple bioinformatics calculations on a specific protein sequence - and for
Useful Protein Molecular Weight Calculator kDa
Protein molecular weight calculator kDa The rule of thumb for conversion of microgram amounts into picomole amounts at different protein molecular weights and peptide molecular weights is: 1000/Mw of protein in kDa = pmol/ug You can use the table below for a quick conversion of your protein, depending on its size: Protein size kDaMicrogramPicomoleMicrogramPicomole1110001100010101000110020201000150505010001201001001000110150150100016.7 In other situations you may want to learn more about the
Latest in: Database Searching & Protein Identification See all
Speedy and detailed protein ID of SDS gel bands
Question: Can I identify hundreds of gel bands in a few days? The biopharmaceutical company that I work for, consistently uses SDS PAGE and Western Blotting to check protein expression. But this means we spend a great deal of money on antibodies, which I feel may be unnecessary. In addition, there are several unknown proteins in the gels, possibly degradation products, which
GPMAW lite – Protein Sequence Calculator (and much more)
When working with proteins you often need to make simple bioinformatics calculations on a specific protein sequence - and for
Common protein contaminants in mass spectrometric protein ID
Questions: Why was Keratin identified in my 1D gel band? Is the keratin determined by the protein identification service in my
Latest in: mAb analysis See all
Critical reasons why mAbs call for orthogonal HCP analysis techniques
Question: HCP-ELISAs show our mAb is pure. So why do we need orthogonal analysis? We produce a monoclonal antibody (mAb) product,
Investigation of ubiquitin in purified antibody products
Question: Should we worry about ubiquitin in our mAb? Our process-specific HCP ELISA did not detect any Host Cell Proteins (HCPs) in our purified monoclonal antibody (mAb). However, mass spectrometry-based HCP analysis identified ubiquitin in low-ppm amounts. Do you have any suggestions for why the Protein A purification did not remove it? Why did ELISA not detect it? And should we worry about
Assessing and monitoring the quality of therapeutic mAb products
Question: Which analyses should be part of mAb characterization? I need to ensure the quality and consistency of my mAbs. Which analyses
Latest in: MW Analysis of Intact Proteins See all
Buffers for MALDI mass spectrometry analysis of a protein
Question: What buffer should I use for MALDI analysis? What buffer should I use for MALDI Mass Spectrometry analysis of my protein sample? Answer: In general, MALDI-MS is more tolerant to contamination than other MS methods. However, the salts and detergents that are commonly used in biomolecular science can still interfere. Either with crystallisation or ionisation or both. Therefore particular attention should be paid to sample