µPAC in Practice – Part I
By Pat Sandra, Emeritus Professor, Organic Chemistry, Ghent University; Founder and President, Research Institute for Chromatography, Kortrijk, Belgium.
It is well documented that miniaturization of the separation medium offers several advantages for high-resolution LC-MS analysis of complex samples, such as those encountered in proteomics, metabolomics, and biopharmaceutical characterization. The inherent high permeability and low on-column dispersion obtained by the perfect order of the separation bed in micro-pillar array columns (µPAC) is superior to packed beds and monoliths for high-resolution separations. We have recently illustrated this in a number of applications using the first generation of commercially available µPAC to analyze biopharmaceuticals using µPAC combined with mass spectrometry (µPAC-MS), described below. Moreover, the µPAC concept can in the future be tuned and optimized for production of separation media with unique features for high throughput and high productivity applications as well … to follow!
Characterizing antibody-drug conjugates
Kadcyla (ado-trastuzumab emtansine) and, for comparison, Herceptin (trastuzumab) tryptic digests were analyzed on a 200 cm µPAC C18 column and peaks eluting were detected by UV spectroscopy and high-resolution MS. The resolving power of the µPAC column allows an in-depth study of conjugation sites and the separation of isomeric conjugated peptides.
Figure 4. µPAC-UV 214 nm peptide map of Herceptin and Kadcyla.
Figure 5. µPAC-UV 214 nm peptide map of a Remicade originator (A) and candidate biosimilar (B) tryptic digest. Unzoomed and zoomed views.
Monoclonal antibody peptide mapping
Remicade (infliximab) originator and candidate biosimilar tryptic digests were analyzed on a 200 cm µPAC C18 column and peaks eluting were detected by UV spectroscopy and high-resolution MS. The simultaneously acquired MS and MS/MS data enabled us to identify differences between the originator and candidate that rule out the candidate for further development.
µPAC in Practice – Part II
An interview with Goran Mitulović, Head of the Proteomics Core Facility at the Medical University of Vienna, Austria.
You have a great deal of experience with nano LC...
I have been applying capillary and nano HPLC since 1999, first as a user and later as a researcher at LC Packings – the pioneers of nano HPLC. My first analysis was of alpha-amanitin in the laboratory of Rainer W. Schmid at the Medical School in Vienna (now the Medical University of Vienna) – I remember being excited about the sensitivity. Later, after I joined LC Packings, I addressed more complex separation problems and became very intimate with nano HPLC.
What did you think when you first heard about µPAC technology?
Actually, I first heard about “pillar array columns” at the 2013 HPLC conference in Amsterdam from Wim De Malsche. I was curious, but they were not commercially available at that time. It was Jean-Pierre Chervet who directed my attention to µPAC at the 2017 ASMS meeting in Indianapolis. After that conversation, I checked the PharmaFluidics web page and ordered my first column!
What was your first experience with µPAC like?
I already have experience with monolithic columns and so I was expecting something similar. However, my first separation runs did not go as expected – until I realized that the void volume of the column is different and requires a different approach to a classical nano column! Afterwards, once I partially optimized injection and the separation gradient, everything went smoothly. And I must say that this all happened within three hours of the first installation of the column. I know that PharmaFluidics advises using the column for direct injection; however, I’ve been using them with a trap column and have not observed any loss of separation performance.
What applications are you working on with µPAC?
Our main application is the separation of peptides originating from biological samples. Analyses include simple mapping, identification of post-translational modifications and characterization of biosimilars. A major research question looming on the horizon is the analysis of therapeutic antibodies, which I am starting to investigate.
What does µPAC enable you to do differently?
The peak capacity of µPAC columns is significantly higher than the peak capacity of conventional packed columns, leading to a higher number of identifications and, more importantly, better sequence coverage. The low backpressure generated by these columns and the ability to “reverse flush” them add to the benefits. As I mentioned, I use a trap column in front of the µPAC, which removes the majority of possible contaminants; however, when using the same setup with a packed column, I often experienced blockages. So far, the µPAC has been running 24/7 since the beginning of October. There have been no blockages, or pressure increases. Such robustness is surprising!
I’ve also been impressed by the excellent reproducibility, even after repeated injections of extremely complex samples, such as proteins from bone, saliva, urine, serum, and cell cultures.
What does the future hold for µPAC?
µPAC technology will have a bright future. So far, only C18 chemistry is available, and I would strongly encourage PharmaFluidics to develop new chemistries; for example, HILIC, which is truly complementary to C18 phase separations.
I also envisage that by manufacturing shorter columns (200–250 mm), µPAC could be used in clinical analysis, which would be a real revolution: fast flow, low backpressure, excellent robustness.
Pat Sandra is Emeritus Professor of Organic Chemistry at Ghent University, and Founder and President of the Research Institute for Chromatography (RIC), Kortrijk, Belgium. “Through the activities of RIC, I got in touch with the real analytical needs of the industry and found we could help in providing solutions that are economically relevant. Moreover, it allowed me to keep my best PhD students around me, which resulted in high scientific output in a non-academic environment,” he says.
