Assessing and monitoring the quality of therapeutic mAb products
Question: I need to ensure the quality and consistency of my mAbs. Which analyses should be conducted 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 drug approved from 2015 to 2018 were mAbs [2]. Also, the […]
Latest in: Analysis of Host Cell Protein See all
Why measure process-related impurities in cell and gene therapies with mass spectrometry?
Question: I work in a biopharmaceutical company developing a gene therapy product based on an adenovirus vector. Recently, I have become
Can I use commercial HCP ELISA kits for my clinical trial application?
Question: My company uses a commercial HCP ELISA kit for host cell protein analysis of clinical phase 1 material. We hear
Latest in: Analysis of Biopharmaceuticals See all
Speedy and detailed protein ID of SDS gel bands
Question: 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 we would like to identify. Is there a fast and reproducible
Assessing and monitoring the quality of therapeutic mAb products
Question: I need to ensure the quality and consistency of my mAbs. Which analyses should be conducted for proper mAb characterization? Answer: For
How to characterize antibody glycosylation by HILIC-MS
Question: 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 linkage site, how the glycan is branched,
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: I found that the N-terminal of my protein is blocked. How can I then analyze it by Edman degradation / n-terminal sequencing - and what is causing the problems? Answer: N-terminal sequencing is a requirement according to the ICH Q6B Guideline. This includes structural characterization of recombinant proteins for clinical testing, and to demonstrate comparability and consistency between cGMP batches [1]. It is
3 Techniques for finding protein N- and C-terminal sequence
Question: Where does the amino acid sequence start and where does it end? Can you describe relevant sequencing techniques from the
Preparing samples for N- and C-terminal sequencing by MALDI ISD
Question: 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 so we can confirm the sequence of the
Latest in: Amino Acid Analysis See all
Why you need accurate concentration determination of protein standards
Question: 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 laboratories. They are based on UV
Determine the Molar Extinction Coefficient of protein in 3 small steps
Question: I have a purified protein that I would like to quantify accurately in my lab using 280 nm UV measurement
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: 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 we would like to identify. Is there a fast and reproducible
1D SDS PAGE versus 2D PAGE. Any recommendations?
Question: I’m interested in isolating and identifying cell surface receptor proteins. They typically have high MW, are glycosylated, and have transmembrane domains. I’m
How much protein should I load for protein identification by mass spec
Question: 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) and cause spot streaking in the gel [1,2]. A
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 kDaThe 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: 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 we would like to identify. Is there a fast and reproducible
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: Is the keratin identified by the protein identification service in my 1D gel band a contamination? Or is it
Latest in: mAb analysis See all
Assessing and monitoring the quality of therapeutic mAb products
Question: I need to ensure the quality and consistency of my mAbs. Which analyses should be conducted for proper mAb characterization? Answer: For
How to characterize antibody glycosylation by HILIC-MS
Question: 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 linkage site, how the glycan is branched,
4 reasons to perform N-terminal protein sequence analysis by Edman
Question: How do I obtain the N-terminal sequences of the heavy chain and light chain of my monoclonal antibody? Is there a prefered protein sequence analysis method for mAbs? Answer: mAb N-terminal sequence analysis is often performed using high resolution mass spectrometry for peptide mapping by LC MS/MS [1]. However, sequencing by the classical Edman degradation offers some advantages over LC MS peptide mapping
Latest in: MW Analysis of Intact Proteins See all
What buffer to use for MALDI mass spectrometry analysis of a protein?
Question: 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 purification and storage solvents. Do not utilise