Chapter 2 described the development of a gel‐based analytical platform to
independently monitor both the PEG and protein components of a PEGylated protein;
thereby providing a measure of the disposition, pharmacokinetics and structural integrity
of the conjugate in a rodent model, and could potentially be translated to the analysis of
samples generated from clinical studies. Here, a 1H NMR method was developed and
optimised for the detection and quantification of PEGylated proteins, and was used to
quantify the amount of 40KPEG‐insulin excreted in urine from rodents from the 28 day
disposition study; thus adding an extra facet to the gel‐based platform described in chapter
2 in terms of absolute quantification, since the gel‐based methods provide only semi‐
quantitative information. PEG contains many hydrogen atoms each in the same chemical
environment, which all contribute toward a single resonance peak that can be easily
identified. The straight‐forward identification of PEG by 1H NMR potentially represents a
facile, uncomplicated tool to monitor the PEG moiety of PEGylated proteins. However,
since the area under the curve of each resonance signal is directly proportional to the
number of protons which produce the signal, this reduces the ability of 1H NMR to
simultaneously monitor the insulin component of the conjugate, as, in comparison to PEG,
the numbers of hydrogen atoms present in insulin are significantly lower. However, this
does not preclude the use of 1H NMR as an effective tool to quantify PEG excreted in urine.
Indeed, with 1H NMR a sealed IS could be used to produce a fully quantitative assay,
allowing for robust analysis between samples. In this study, nicotinamide was selected as
the IS, as it is non‐labile, non‐volatile, and resonates downfield from the PEG resonance.
Urine samples from the 28 day disposition study were prepared for 1H NMR analysis as they
were for gel‐based analysis, by dialysis and deproteination, which was shown to
reproducibly remove abundant urinary proteins in gel‐based analyses. The presence of
dialysis and deproteination were again used to clean up urine samples prior to analysis.
This kept sample preparation identical between both gel‐based and 1H NMR platforms,
ensuring reliable comparisons could be made between the data generated by each assay.
Prior to analysing urine samples from the 28 day disposition study, the various
spectrometer parameters involved in producing 1H NMR spectra were optimised. This was
crucial to ensure accurate and reliable quantification of PEG could be maintained during
analysis of each sample. Once these parameters were defined, they were used throughout
for all subsequent analyses of urine samples collected during the 28 day disposition study.
1H NMR analysis confirmed that 40KPEG‐insulin was excreted rapidly in the urine from day 1 to day 3, after which the amount excreted remained relatively stable throughout the
remaining time points, with slight increases in PEG excreted on days 4 and 17. The
sensitivity of 1H NMR, under the conditions used in this study, was sufficient to detect PEG
in all urine samples analysed, with the LOD and LOQ defined as 0.5 µg/mL and 10 µg/mL,
respectively, but could not provide full quantification after day 3, when the amounts of PEG
excreted fell below the LOQ. Despite this, quantification of PEG by 1H NMR was
commensurate with the values obtained using gel‐based methods. Using a Bruker 600 MHz
spectrometer, 1H NMR was not sensitive enough to monitor the protein moiety of the
conjugate. Future advancements in NMR technology, coupled with the use of more
powerful spectrometers, such as a Bruker 800 MHz or Bruker 1 GHz spectrometer, may
result in the capability to simultaneously monitor each component of a PEGylated protein
using 1H NMR. Increased sensitivity may also allow the detection and quantification of PEG
in other biological fluids and tissues: urine was selected for initial 1H NMR analysis due to
the relative ease in concentrating urine samples; future increases in sensitivity may
therefore preclude the necessity of sample concentration and allow for detection of PEG in
other samples, such as plasma. Furthermore, when translated to clinical studies,
urine typically has a much lower protein content; hence, there may be much less analyte
loss during sample preparation and consequently this may improve the sensitivity of the
assay. Thus, the conclusion from this study was that 1H NMR can be used as a tool to
measure the urinary excretion of PEG, with benefits such as high specificity, since PEG
produces a single resonance peak, and absolute quantification, using an internal standard.
Coupled with the gel‐based analytical platform, 1H NMR provides complementary
information with regards to assessing the pharmacokinetics, disposition and biological fate
of PEGylated proteins; with similar analysis parameters to the gel‐based assays, such as
identical LOD, sample preparation and run‐time, as well as offering the benefit of absolute
quantification of the PEG moiety – as detailed in table 3.2.
Gel‐Based Platform 1H NMR
LOD 0.5 µg/mL 0.5 µg/mL
Structural Analysis PEG, Linker, Protein PEG
Sample Volume 100 – 300 µL 1 mL
Sample Preparation Dialysis and deproteination Dialysis and deproteination
Overall Time 2 days 2 days
Analysis Requirements Routine laboratory
equipment
Specialist equipment
Quantification Semi Absolute
Table 3.2 Comparison of the gel‐based analytical platform and 1H NMR