Raman Spectroscopy to Monitor Post-Translational Modifications and Degradation in Monoclonal Antibody Therapeutics

Monoclonal antibodies (mAbs) represent a rapidly expanding market for biotherapeutics. Structural changes in the mAb can lead to unwanted immunogenicity, reduced efficacy, and loss of material during production. The pharmaceutical sector requires new protein characterization tools that are fast, applicable in situ and to the manufacturing process. Raman has been highlighted as a technique to suit this application as it is information-rich, minimally invasive, insensitive to water background and requires little to no sample preparation. This study investigates the applicability of Raman to detect Post-Translational Modifications (PTMs) and degradation seen in mAbs. IgG4 molecules have been incubated under a range of conditions known to result in degradation of the therapeutic including varied pH, temperature, agitation, photo, and chemical stresses. Aggregation was measured using size-exclusion chromatography, and PTM levels were calculated using peptide mapping. By combining principal component analysis (PCA) with Raman spectroscopy and circular dichroism (CD) spectroscopy structural analysis we were able to separate proteins based on PTMs and degradation. Furthermore, by identifying key bands that lead to the PCA separation we could correlate spectral peaks to specific PTMs. In particular, we have identified a peak which exhibits a shift in samples with higher levels of Trp oxidation. Through separation of IgG4 aggregates, by size, we have shown a linear correlation between peak wavenumbers of specific functional groups and the amount of aggregate present. We therefore demonstrate the capability for Raman spectroscopy to be used as an analytical tool to measure degradation and PTMs in-line with therapeutic production.


Figure S1
Population (%) of monomers, aggregates and fragments in the IgG4 samples after degradation determined using SE-UPLC.

Figure S4
Oxidation and deamidation PTMs determined by peptide mapping.

Table S1
Summary of SE-UPLC results as a percentage of aggregate, monomer and fragments species present in degraded sample.

Table S2
Detailed peptide mapping results reporting the extent of PTM resulting in oxidation highlighting peptide fragments involved. Where HC is the heavy chain and LC is the light chain.

Table S3
Detailed peptide mapping results reporting the extent of PTM resulting in deamidation highlighting peptide fragments involved. Where HC is the heavy chain and LC is the light chain.

Figure S5
Raman spectra under all degradation conditions: (A) raw Raman data for each degradation condition, (B) Buffer subtracted Raman data and (C) Fully preprocessed Raman data for each condition where the pre-processing includes baseline correction, smoothing and normalisation as described in supplementary methods above.

Figure S6
Raman spectrum of NaP 20 mM and NaF 150 mM at pH 6 analysis buffer with peak assignments. Data shown are an average of 8 spectra with no preprocessing used for buffer subtraction in Figure 5 B.

Table S4
Summary of the Trp peak wavenumber as an average of 8 repeats with calculated standard errors (SE). Results are compared in the buffer subtracted and data without buffer subtraction.

Figure S7
Colour change of degraded products assigned to the oxidation species of the Trp. IgG4 at 25 mg mL -1 in formulation buffer.

Table S5
Detailed peptide mapping results reporting the extent of PTM resulting in glycation.

Figure S8
Raman spectra of chemical standards of Trp and its degradation products Kyn, NFK and Trp-OH all dried onto to steel slides. Spectra were pre-processed using asymmetric least squares only baseline correction.

Figure S9
SEC trace of degraded IgG4 under UV light (5000 kLux.h) at 45 mg mL -1 in formulation buffer.

Figure S11
SE-UPLC analysis of each of the separated aggregate fractions. Run at 1 mg mL -1 in formulation buffer.

Figure S12
Raman spectra of formulation buffer undergoing the same 5000 kLux.h incubation with peak assignments. Data shown are averages of 8 spectra with no pre-processing. Average spectrum was used for buffer subtraction in Figure  S13B. Numbers on the spectra refer to wavenumbers (cm -1 ).

Figure S13
Raman data analysis of all SEC separated and concentrated aggregate fractions: (A) Raw Raman data for each fraction, (B) Buffer subtracted Raman data and (C) Fully pre-processed Raman data for fraction where the preprocessing includes baseline correction, smoothing and normalisation as described in supplementary methods above.

Figure S14
Average peak centres of the a) C-N, b) CH2, CH3 deformation and c) Trp indole N-H vibration from 8 Raman repeats. Error bars highlight the standard error.

Reagents and materials
All chemical reagents were of analytical grade and used with no additional purification unless otherwise stated. IgG4 samples were kindly provided by UCB Pharma, Slough. 5-Hydroxytryptophan (Trp-OH) and kynurenine (Kyn) were purchased from Sigma-Aldrich (Missouri, US). N-formylkynurenine (NFK) was synthesised by BOC Sciences (New York, US). Ammonium bicarbonate BioUltra ≥99.5%, hydrogen peroxide 30% (w/w) in H2O were purchased from Sigma-Aldrich. Ammonium hydroxide solution 28% (NH3 in H20), HCl 37% and Sodium phosphate (NaH2PO4.H2O and Na2HPO4) were purchased from VWR AnalaR Normapur. NaOH Solution 1 M volumetric solution was purchased from Honeywell Fluka Sodium Fluoride analytical reagent grade, DTT and iodoacetamide were all purchased from Thermo Fisher Scientific. Trypsin sequencing grade was obtained from Promega.

SEC Separation of IgG4 Aggregates
Samples were collected from the fractions shown in Figure S3. The fractions were pooled for each SEC peak and concentrated to 20 mg mL -1 using 10 kDa Millipore spin columns. SE-UPLC analysis for these samples was carried out in formulation buffer.

Sample Preparation of IgG4 Degradation Samples
After 14 days, all degradation samples were buffer exchanged from the formulation buffer to sodium phosphate 20 mM, sodium fluoride 150 mM at pH 6. Sodium fluoride was used as fluoride does not absorb in the UV region 180-260 nm for CD whereas chloride does. The final concentration for all samples was adjusted to 25 mg mL -1 . SE-UPLC analysis was carried out after buffer exchange.

SE-UPLC Analysis of Degradation Conditions
SE-UPLC analysis was performed using a Waters Acquity UPLC equipped with a BEH 200 Å, 1.7 µm SEC column. Sodium phosphate 20 mM, sodium fluoride 150 mM at pH 6 was used as the mobile phase with an isocratic flow at 0.35 mL min -1 resulting in a 10 min run time per sample. The eluent was monitored using UV detection at A280 and fluorescence at room temperature. The samples were diluted to 1 mg mL -1 and a load volume of 2 µL was used. Data were anlaysed using Waters Empower using the integral of chromatogram to estimate the quantity of monomer, aggregates and fragments. Analysis of the IgG4 aggregates used the same parameters but remained in the formulation buffer as no CD was carried out.

Peptide Mapping
To prepare samples for tryptic digestion, 80 μg of A33 IgG4 was evaporated and resuspended in 60 μl of 8 M guanidine hydrochloride (Sigma-Aldrich, Gillingham, UK). Two μL of 500 mM dithiothreitol (Thermo Fisher Scientific, Rockford, IL, USA) in 750 mM Tris-HCl buffer pH 7.9 was added to the samples and incubated for 40 minutes at 37 o C. Next, 6 μL of 500 mM iodoacetamide (Thermo Fisher Scientific) in 750 mM Tris-HCl buffer pH 7.9 was added to the samples and incubated for 30 min at room temperature in the dark. Samples were desalted using Zeba spin plates (Thermo Fisher Scientific) according to the manufacturer's instructions. Next, 3.5 µg of sequencing grade modified trypsin (Promega, Madison, WI, USA) in 7.5 mM Tris-HCl buffer (pH 7.9) was added and incubated for 3 hours at 37 o C. Proteolysis was quenched by the addition of 5 µl 1 % trifluoroacetic acid (Sigma-Aldrich).
Peptides were separated using online reversed phase liquid chromatography (Dionex UltiMate 3000), using a binary solvent system consisting of mobile phase A (water ((PURELAB Ultra, ELGA, High Wycombe, UK))/0.1% formic acid (Thermo Fisher Scientific,)) and mobile phase B (acetonitrile (ROMIL, Cambridge, UK)/ 0.1% formic acid (Thermo Fisher Scientific)). Peptides were loaded onto an ACQUITY UPLC BEH C18 Column (130 Å, 1.7 µm, 2.1 mm X 50 mm) (Waters, Milford, MA, USA). Peptides were injected in mobile phase A and separated over a linear gradient from 1 % to 36 % mobile phase B with a flow rate of 200 µL/min. Samples eluted directly via a heated electrospray source into the mass spectrometer.

SEC Separation of IgG4 Aggregates
Aggregates were purified from monomer using a HiLoad 26/600 Superdex 200 pg column on an Akta purifier system. Samples were collected from the fractions shown in Figure S9. Figure  S10 shows the composition of the fractions, determined using an SDS page gel and SE-UPLC ( Figure S11). IgG4 sample aggregates can be broken into heavy and light chains by using reducing conditions. The fractions were pooled for each SEC peak and concentrated to 20 mg mL -1 .

Circular Dichroism Spectroscopy
Circular dichroism analysis was carried out using a Chirascan-plus qCD (Applied Photophysics Manchester, UK). A quartz cuvette with a path length of 0.2 mm was used to collect scans between 180 -260 nm for far UV CD. For near UV CD a path length of 1 cm was used with scans collected between 250 -320 nm. All spectra were collected at 20 °C. Each sample was aliquoted into 3 wells, with 3 repeats taken per well. The data were averaged for each well and converted into mean residue ellipticity (MRE) [θ] using Equation 1 below. Averaging for each well negated the use of pre-processing methods such as data smoothing.

Eq. 1
Where θ is the CD signal in millidegrees, L is the pathlength in cm of the cuvette and C is concentration of the protein in mg mL -1 . The exact concentration was calculated form A280 using the near UV absorbance spectrum for each sample using the extinction coefficient. The mean residual weight (MRW) is MW/ (n-1) where MW is the molecular weight in Daltons and n-1 is the number of peptide bonds. The parameters used were 1 s acquisition, 0.5 nm step, bandwidth 1.0 nm and 20 °C.

Raman Spectroscopy
A Renishaw inVia 785 nm Raman microscope (Renishaw Plc., Gloucestershire, UK) was calibrated using a x50 objective, 10% power and 1 s acquisition focused onto a silicon plate centered at 520 cm -1 .
The experimental parameters were a laser power of ~30 mW on a sample, 10 s acquisition, 20 accumulations, 600 l/mm centered at 1500 cm -1 . These parameters resulted in an overall acquisition time of 200 s per measurement. A 15 mm long distance objective (Renishaw) was used. The samples were aliquoted into a 96 well quartz plate, previously calibrated for automated plate mapping. The wells hold 360 μL of solution. To account for laser power drift and well to well variation the samples were randomised and two well repeats of each sample were used. 4 repeats were then taken from each well resulting in 8 repeats overall per sample. Raw and pre-processed spectra are shown in Figure S5 and S10. For the spectra in Figure 3a and 5a, the 8 repeats were averaged for each sample. For the PCA, all repeat data were analysed separately to show the well to well variation, as well as within well variation for the 96 well plate set-up.
Trp chemical standards were suspended in MilliQ water and 2 μL was spotted and dried onto steel plates. The dried samples were analysed using x50 objective (Renishaw), 50% laser power, 600 l/mm, centered at 1500 cm -1 , 1 s acquisition, 5 accumulations with 3 repeats per sample. The spectra are shown as an average of 3 measurements.

Analysis and Data Processing
All data pre-processing and subsequent analysis was performed within Matlab R2018a using in-house toolboxes. For data processing the baseline was subtracted from every sample. For the degradation samples the buffer subtracted was sodium phosphate 20 mM, sodium fluoride 150 mM at pH 6. For the IgG4 SEC separated aggregates the buffer used was the formulation that had also undergone 5000 kLux.h incubation. The data were then baseline corrected using asymmetric least squares (ASL) and smoothed using Gaussian moving windows with a window width of 4 [1,2]. The data were then normalised uses standard normal variate (SNV) and the wavenumbers not of interest were discarded.

Figure S1
Population (%) of monomers, aggregates and fragments in the IgG4 samples after degradation determined using SE-UPLC.

Figure S4
Oxidation and deamidation PTMs determined by peptide mapping. Results shown are calculated as the % that at least one of the amino acids in the antibody had a particular PTM. Sequence coverage was reported to be 99.1% with an average mass error of ±1.2 ppm compared to the expected mass of each peptide fragment for the HC. The sequence coverage for the LC was reported to be 100% with an average mass error of ±1.1 ppm.
These percentages correspond to the probability that at least one modification of this type has occurred on the A33 antibody (2xheavy and 2xlight chains) and are given by:

Table S4
Detailed peptide mapping results reporting the extent of PTM resulting in oxidation highlighting peptide fragments involved. Where HC is the heavy chain and LC is the light chain.

Table S3
Detailed peptide mapping results reporting the extent of PTM resulting in deamidation highlighting peptide fragments involved. Where HC is the heavy chain and LC is the light chain.

Figure S5
Raman spectra under all degradation conditions: (A) raw Raman data for each degradation condition, (B) Buffer subtracted Raman data and (C) Fully preprocessed Raman data for each condition where the pre-processing includes baseline correction, smoothing and normalisation as described in supplementary methods above.

Figure S6
Raman spectrum of NaP 20 mM and NaF 150 mM at pH 6 analysis buffer with peak assignments. Data shown are an average of 8 spectra with no preprocessing used for buffer subtraction in Figure S5 B).

Figure S7
Colour change of degraded products assigned to the oxidation species of the Trp. IgG4 at 25 mg mL -1 in formulation buffer.

Table S5
Detailed peptide mapping results reporting the extent of PTM resulting in glycation.

Figure S8
Raman spectra of chemical standards of Trp and its degradation products Kyn, NFK and Trp-OH all dried onto to steel slides. Spectra were pre-processed using asymmetric least squares only baseline correction.

Figure S9
SEC trace of degraded IgG4 under UV light (5000 kLux.h) at 45 mg mL -1 in formulation buffer.

Figure S11
SE-UPLC analysis of each of the separated aggregate fractions. Run at 1 mg mL -1 in formulation buffer.

Figure S12
Raman spectra of formulation buffer undergoing the same 5000 kLux.h incubation with peak assignments. Data shown are averages of 8 spectra with no pre-processing. Average spectrum was used for buffer subtraction in Figure  S13B. Numbers on the spectra refer to wavenumbers (cm -1 ).

Figure S10
Raman data analysis of all SEC separated and concentrated aggregate fractions: (A) Raw Raman data for each fraction, (B) Buffer subtracted Raman data and (C) Fully pre-processed Raman data for fraction where the preprocessing includes baseline correction, smoothing and normalisation as described in supplementary methods above.

Figure S14
Average peak centres of the a) C-N, b) CH2, CH3 deformation and c) Trp indole N-H vibration from 8 Raman repeats. Error bars highlight the standard error.