Ultrahigh-pressure liquid chromatography (UHPLC) is very useful for four key methods used in biotechnology: peptide mapping,
amino acid analysis, intact protein analysis, and glycoprofiling. The first two methods are covered here.
Biopharmaceuticals have become the major products of the biotechnology industry around the world today (1–4). Biopharmaceuticals
include recombinantly derived proteins, peptides and or antibodies, with glycoproteins and antibodies being the most common
and perhaps most important (5–9). Meeting the requirements of the US Food and Drug Administration or other regulatory bodies
for such products involves a demonstration of drug substance characterization (as fully as possible), lot-to-lot and batch-to-batch
comparisons, stability studies, impurity profiling, glycoprofiling, and numerous other analytical studies (8), all of which
can take years to derive, interpret, and profile in the final Chemistry, Manufacturing and Controls documents (CMC). (And
all that information is separate from clinical safety and efficacy studies).
In this and subsequent columns, we endeavor to describe the most important and useful analytical methods used in the biopharmaceutical
industry to accomplish the above mentioned studies, and especially to meet CMC documentation for regulatory submittals. Clearly,
conventional high performance liquid chromatography (HPLC) and mass spectrometry (MS) have evolved to become perhaps the most
informative and popular techniques in routine usage today to meet these goals. However, as this column will describe, both
of these methods have and will continue to evolve and improve. HPLC is now being replaced by ultrahigh-pressure liquid chromatography
(UHPLC). MS also is evolving, in both the hardware and software. Instruments provide better mass accuracy, resolution, and
sensitivity with increased ruggedness and ease of use. Software enhancements provide powerful tools for interpreting the very
large body of data generated in characterizing a protein pharmaceutical. It is virtually impossible in today's biotech world
to meet regulatory requirements without the heavy use of these two, now totally complementary analytical methods and instrumentation,
combining them with electrospray ionization (ESI) in UHPLC–ESI-MS.
The examples in this column will use Waters instrumentation and methods, but we hasten to indicate that much or most of that
which follows is entirely possible and feasible on other vendors' UHPLC instrumentation, with some caveats. The pressure limit
of some chromatography instruments is not compatible with the high back pressures commonly encountered in UHPLC. To some extent,
raising the temperature can obviate this restriction by reducing the viscosity and, therefore, back pressure. When following
that approach, users must monitor changes in selectivity with temperature. Of course, by reducing the system pressure, one
does not fully exploit the promise of UHPLC. In any case, the essence of UHPLC is improving resolution and sensitivity by
reducing dispersion. An increase in throughput often accompanies these benefits.