Determination of glycation levels in Erwinia chrysanthemi asparaginase drug product by liquid chromatography – mass spectrometry

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Determination of glycation levels in Erwinia chrysanthemi asparaginase Drug Product by liquid chromatography – mass spectrometry Patrick Kanda , Thomas T. Minshull PII: DOI: Reference:

S0928-0987(20)30042-7 https://doi.org/10.1016/j.ejps.2020.105253 PHASCI 105253

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

20 September 2019 9 January 2020 1 February 2020

Please cite this article as: Patrick Kanda , Thomas T. Minshull , Determination of glycation levels in Erwinia chrysanthemi asparaginase Drug Product by liquid chromatography – mass spectrometry, European Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.ejps.2020.105253

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Highlights   

Erwinase glycation levels can be quantified by mass spectrometry. Erwinase becomes glycated following formulation with glucose. Glycation of Erwinase Drug Product in lyophilized form continues at low temperature.

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Title: Determination of glycation levels in Erwinia chrysanthemi asparaginase Drug Product by liquid chromatography – mass spectrometry Authors: Patrick Kanda a and Thomas T. Minshull

Address:

a

b

Porton Biopharma, Ltd. Manor Farm Road Porton Down Salisbury SP4 0JG United Kingdom

Corresponding author.

E-mail address: [email protected] Phone: +44 (0)1980 551710 b

Present address: Immunocore Limited 101 Park Drive Milton Park Abingdon Oxon OX14 4RY

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Abstract Erwinase (Erwinia chrysanthemi L-asparaginase) Drug Product (DP) is a freezedried formulation with a three-year shelf life at 2-8 o C, and an established safety, stability and efficacy profile over the more than three decades of clinical use. Seven Erwinase® DP batches, released over a 7-year period, were screened by reversedphase liquid chromatography coupled to time-of-flight mass spectrometry for glycation levels. This modification is a known and natural consequence of exposure of Erwinase Drug Product to glucose excipients in stabilizing formulations. Although glycation is detected in current release and stability methods, glycation, including the conditions under which this reaction occurs, has not been previously characterised in detail. We have found that glycation levels of different DP lots generally correlated with age, when they were stored at low temperature. This suggests that the glycation reaction continues over time within the Drug Product formulation in the lyophilised state, even under low temperature (+2 - 8oC) conditions. We were also able to examine glycation levels of one DP lot, Lot D, held under long term stability at 3 different temperatures over a 5-year period. The 2 samples held at -20o C and -80o C, were glycated to levels of 12% and 17%, respectively. However, the DP Lot D sample held at +2 – 8o C in this time period was found to be glycated to a level of 35.6%, with multiple glycations of individual subunits observed. For analytical reference materials, it is important to keep parameters such as glycation levels as constant as possible, to avoid a ‗moving target‘ with respect to comparisons with release and stability testing. These data suggest that storage of DP as reference standards at a lower temperature (e.g., -20o C) can significantly reduce levels of glycation over the longer time periods required for analytical reference standards.

1.

Introduction

The manufacture of therapeutic proteins via fermentation/expression systems generally results in a protein Drug Substance (DS) which must be stabilised against degradation and aggregation during its shelf life as a Drug Product (DP). One of the most effective ways to achieve this involves formulating the protein with excipient stabilisers such as saccharides (Arakawa and Timasheff, 1982; Carpenter et al., 1987). An aqueous mixture is then dispensed into individual dose vials, followed by lyophilisation under sterile conditions. For many such proteins, low temperature (~ +4oC) storage of properly sealed aliquots of freezedried DP, under controlled conditions, confers a useful shelf life measured in months and years, thus facilitating their distribution and administration to patients in geographically diverse markets. Porton Biopharma Limited (PBL), previously the biopharmaceutical manufacturing arm of Public Health England (PHE), was established in 2015 and is the sole manufacturer of the enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Erwinia chrysanthemi (Erwinase® or Erwinaze®) for the treatment of childhood acute lymphoblastic leukemia (ALL). It is marketed in over 50 countries world-wide as part of a chemotherapeutic treatment programme, and is primarily given to those patients who have been treated first with the Escherichia coli (E. coli) version of this enzyme, but have developed a hypersensitivity reaction to it. Erwinase, which is antigenically distinct from the E. coli asparaginase, is then administered to complete the treatment regimen. No therapeutic alternatives are available to such patients, thus Erwinase is regarded as a life-saving drug.

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Abbreviations: DP DS EDS LC-MS Glc API RP-LC TOF-MS UPLC TFA AGW ACN UV TIC ESI cIEF pI

Drug Product Drug Substance Erwinase Drug Substance Liquid Chromatography – Mass Spectrometry Glucose Active Pharmaceutical Ingredients Reversed Phase - Liquid Chromatography Time-of-Flight Mass Spectrometry Ultraperformance Liquid Chromatography Trifluoroacetic Acid Analytical Grade Water Acetonitrile Ultraviolet Total Ion Current Electrospray Ionisation Capillary isoelectric focussing isoelectric point

The current Erwinase Drug Product (DP) final, freeze-dried formulation has an established safety, stability and efficacy profile over the more than three decades of clinical use, incorporating the entirety of the three-year DP shelf-life. The DP contains approximately 5 mg of D-glucose monohydrate, along with approximately 10 mg Erwinase itself per vial. Sugars, including monosaccharides such as glucose monohydrate, have been used as protein stabilisers and lyoprotectants for many years (Arakawa and Timasheff, 1982; Carpenter et al., 1987). Erwinase is a tetramer composed of 4 identical subunits. At appropriate levels, glucose monohydrate promotes stability of the reconstituted DP by preventing lyophilisation-induced dissociation of the protein into its subunits, as well as minimising its aggregation (Hellman et al., 1983). Other sugars have likewise been demonstrated to perform a similar function for Erwinase at about the same efficiency as glucose (Ward et al., 1999). Glucose monohydrate also helps control the moisture content of the lyophilised final product (TreDenick, 2008). The use of saccharides as excipients often leads to unwanted glycation of Active Pharmaceutical Ingredients (API) (Fischer et al., 2008; Vrdoljak et al., 2004; Quan et al., 1999). The risk of glycation is increased when reducing sugars, such as glucose, are included in the final formulation (Andya et al., 1999; Zheng et al., 2006; Gadgil et al., 2007). With proteins such as Erwinase, the glycation event generally involves nucleophilic attack of a primary ε-amino group of lysine or the N terminus on the carbonyl carbon of glucose in the Maillard reaction to form an initial Schiff base (Fig. 1) (Maillard, 1912). It should be noted that within a DP dose in a given vial, glucose is present at a 5-6 fold molar ratio to available lysines in Erwinase. The N-substituted glycosamine undergoes an Amadori rearrangement to give the more stable ketoamine derivative (Amadori product), resulting in a MW increase of about 162 mu (Zhang et al., 2009). Previous studies on the effects of protein glycation have shown impact on charge profile (Quan et al., 2008), antibody-antigen interactions (Kennedy et al., 1994), and protein structure and stability (Luthra and Balasubramanian, 1993). These perturbations in protein structure/function may affect product efficacy (in terms of both potency and

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pharmacokinetics) and safety (immunogenicity). The major class of therapeutic proteins in which these observations have been made are immunoglobulins (IgGs), both recombinantexpressed monoclonal IgGs and pooled, plasma-purified polyclonal products (Fischer et al., 2008; Leblanc et al., 2016). In reviewing the historical role(s) protein glycation plays in human physiology and metabolism, the most attention has been given to plasma protein glycation leading to the formation of ―advanced glycation end-products‖ (AGEs). Many of these contribute to the development of chronic vascular complications associated with diabetes, non-diabetic nephropathy, vascular disease, Alzheimer‘s disease, and aging (Ulrich and Cerami, 2001; Kassaar et al., 2017). In contrast, the consequences of relatively short-term exposure of patients to glycated biotherapeutic agents may be more immediate. Glycation in general can occur posttranslationally during a) the upstream manufacturing process via exposure to cell culture feeding sugars (for cellular-expressed products), and b) downstream during purification, formulation with excipients, storage, and post-administration exposure of IgGs to physiological conditions. The bulk of studies reporting the impact of protein glycation focus on intravenous Ig formulations (IVIg) containing reducing sugars, such as glucose, as stabilisers (Fischer et al., 2008; Vrdoljak et al., 2004; Leblanc et al., 2016). These studies employed LC-MS (Fischer et al., 2008; Leblanc et al., 2016; Bozhinov et al., 2010) and 14CGlucose labelling (Vrdoljak et al., 2004) as analytical techniques. The conclusions are mixed regarding glycation‘s impact on Ig stability, immunogenicity, and clearance, even at elevated levels (Leblanc et al., 2016; Yang et al., 2014). However, it has also been observed that glycation levels can be significant, even with formulations kept at low temperature (+2 – 8oC) (Fischer et al., 2008; Vrdoljak et al., 2004). These observations were made with Ig intravenous formulations in the liquid state. There have also been prior studies on the glycation of biotherapeutics stored as freeze-dried powders co-lyophilised with reducing sugar excipients under nominal (low temperature) conditions. A recombinant DNase1, formulated with lactose and lyophilised, or as a dry powder for inhalation, underwent glycation with lactose that was both temperature and vial humidity-dependent, with extensive characterisation of the sites of modification by LC-MS of tryptic maps (Quan et al., 1999). Glycation levels of 25% were found at 4 oC after 1 year (Quan et al., 1999). The study examined a single time point of one year at 3 different temperatures. Andya et al. found that glycation levels for an anti-IgE humanised mAb formulated with lactose in spray-dried powders were negligible after 9 months at 5 oC; however, 16 of the 44 lysines were glycated at 30oC during this period (Andya et al., 1999). Again, only a single time point was examined. Zheng et al. found that recombinant interferon-beta-1b (IFN-beta) was glycated by glucose at a level of only 2% following lyophilisation and storage at 4 oC for 2 months, using LC-MS of tryptic maps (Zheng et al., 2006). However, when these same samples were held at 25oC for a further 35 days, glycation levels had increased in proportion to storage time to about 12%, highlighting the influence of temperature on this reaction (Zheng et al., 2006). In the case of Erwinase, the yeast extract (Ubichem, Worcestershire, UK) used as a nutrient source in the fermentation of the Erwinia chrysanthemi (source of Erwinase) contains 13 g carbohydrate/100 g extract, in which mono- and disaccharides are reported to be absent. There are also no sugars or other free aldehydes or glyoxals present during the downstream purification steps. Thus, any glycation of Erwinase is almost solely due to reducing sugar excipients such as glucose introduced during final product formulation. The specific effects of glycation on the quality attributes for Erwinase Drug Product are unknown, though it is clear that glycation is detected during the reversed-phase HPLC release and stability method, and that glycation appears to have no impact on the clinical use of the drug. Erwinase has an established and well-documented safety and efficacy profile spanning decades, using DP across the entire 3-year DP shelf life. Although glycation has been noted in mass spectral analyses of the intact protein following reversed–phase Liquid Chromatography (RP-LC) (data not shown), time-dependent changes in glycation

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levels for specific Drug Product lots following release have not been measured and it is not known how storage conditions impact this. In the current report, we have assessed several Erwinase Drug Product lots by RP-LC linked to Time-of-Flight mass spectrometry (TOF –MS) for glycation and correlated their levels with their lot age since formulation and release, as well as temperature at which stability samples (Lot D) were held. Additionally, some of these lots were also subjected to capillary isoelectric focussing (cIEF) to measure effects of glycation on overall charge.

2.

Material and methods 2.1 Drug Product Lots

The Erwinase Drug Product lots queried in this study were released in a time window from 2010 (Lot C) to 2016 (Lot K). The lyophilised products were all stored in stoppered vials (approx. 10 mg Erwinase/vial, produced under a licensed manufacturing process) and, except for Lot D, were kept at the nominal conditions of +2 - 8oC under minimal light exposure. Lot D has been placed under long term stability at 3 different temperature conditions (-80oC, -20oC, and +2 – 8oC) and a sample vial of each was obtained for this study. Release dates for each lot are given in Table 1. Master Stocks of DP Lots E, F, G, I, and K, at 15mg/ml, were originally prepared as suitable control analytes for unrelated LC methods. They were kept at +2 – 8oC before being diluted into Mobile Phase A for this analysis. DP Lot C was stored lyophilised in vials under nominal conditions (+2 - 8oC) before dissolving in 1 ml of 25 mM sodium phosphate buffer, pH 7, at 10 mg/ml. DP Lot D lyophilised samples, stored under the 3 temperature conditions, were each dissolved in 1 ml of 25 mM sodium phosphate buffer, pH 7.0, to give 10 mg/ml solutions. Fifteen µl of each were diluted into 0.5 ml Mobile Phase A (see Section 2.4 below) for LC-MS analysis. From 1-5 µl of each were subsequently injected onto the RP column. Table 1 Release dates of DP lots analysed by LC-MS for glycation

DP Lot No.

Date Released

1

C

Mar 2010

2

D

Mar 2012

3

E

Nov 2012

4

F

Aug 2014

5

G

Jan 2015

6

I

Nov 2015

7

K

Aug 2016

2.2. Drug Substance Lots Erwinase DS Lots R (45 mg/ml) and S (43 mg/ml) were available as stock solutions in 10 mmol sodium chloride. Five µl of each were diluted into 1 ml Mobile Phase A (see Section 2.4 below) for LC-MS analysis. 2.3. Reagents

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All solvents used for LC–MS were of MS grade from Honeywell (Seelze, Germany) or Sigma (Poole, UK). Trifluoroacetic acid (TFA) (MS Grade, >99%) and formic acid (>99%) were from Fluka. C4 reversed phase columns were from Waters. 2.4. Reversed Phase chromatography-Mass Spectral analysis A Waters Acquity ultraperformance liquid chromatography (UPLC) system was used for the RP chromatographic separation of intact Erwinase DP. The separation method is the licensed, validated reversed-phase (RP) method used for purity assessment in the release and stability of Erwinase DP following manufacture. The column was an Acquity UPLC 2.1 x 150 mm BEH300 C4, 1.7 µm, run at 50oC, using a gradient of 0.1% trifluoroacetic acid (TFA)/Analytical Grade Water, (AGW) (1% Acetonitrile (ACN)), (Mobile Phase A) with increasing amounts of Mobile Phase B (0.08% TFA/ACN, (1% AGW)), at a flow rate of 0.4 ml/min. The ultraviolet (UV) absorbance at 220 nm was monitored. In some cases (DP Lots C, D, G, and DS lots), the gradient was extended (shallowed) somewhat in the region where the protein eluted in an attempt to give better resolution between the main peak and its modified derivatives. This resulted in increases in retention times of the main peaks for these DP lots compared to the original gradient. The UV flow cell was interfaced to a Waters LCT Premier XE Time-of-Flight Mass Spectrometer and the samples subjected to electrospray ionisation (ESI) MS. Total Ion Current (TIC) chromatograms were obtained in the same time window as the UV tracings. The capillary voltage was 3.2 kV, the cone voltage held at 35 V, and the Aperture 1 set at 30 V. Desolvation N2 flow was 900 ltr/hr, the desolvation temperature was set to 350oC, and the source temperature was 110oC. A separate reference probe was interfaced to the source which delivered a leucine-enkephalin lockspray solution as a LockMass to correct for sample mass drift in real time. The spectrum was extracted by a maximum entropy mode (MaxEnt1) program (MassLynx) processing of the TIC chromatogram and deconvoluted to give the spectrum displaying the parent ion masses for the Erwinase subunit and its modified derivatives for the selected chromatogram time window. The raw data was also processed by Waters BiopharmaLynx™ v1.3 software, which also uses MaxEnt1 processing to identify glycated, oxidized, and salt adduct species, as well as tabulate the percent ion total for each. Within this method, a database specifying likely modifications (including mass changes) is applied to identify these variants. Deconvolution parameters can also be manually adjusted to reflect experimental conditions and improve mass accuracy results. 2.5. Capillary isoelectric focussing Analyses of DS and DP samples were performed using a whole-capillary-imaged cIEF system (Model iCE3 with PrinCE autosampler) from ProteinSimple (Toronto, Canada). The isoelectric point (pI) markers used throughout this work were proprietary, small molecular weight, ultraviolet (UV) absorbent markers (pI values 5.85, 6.1, 6.6, 9.5, and 9.77) obtained from ProteinSimple. Samples were prepared immediately before analysis using the following procedure. A master mix (MM) was prepared fresh daily with the composition 8 M urea, 2.2M N-ethylurea, 0.35% methylcellulose, and 4% Pharmalytes 3–10. The analyte samples were prepared using 200 l of MM with 1l of high-pI marker (8.4 or 9.5), 1 l of low-pI marker (4.65 or 5.85), and 1 l of sample (~10 mg/ml). These samples were vortexed briefly to ensure complete mixing and centrifuged at 10,000 rpm for 3 min to remove air bubbles before analysis. Samples were focussed for 1 min at 1.5 kV, followed by 12 to 13 min at 3 kV, and A280 images of the capillary were taken using the ProteinSimple software. The resulting electropherograms were first analysed using iCE3, and pI values were assigned. The data were then downloaded into Empower 2 processing software (Waters, Elstree, UK) for electropherogram integration. Empower data analyses were conducted using Savitsky– Golay smoothing of the electropherogram traces.

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3. Results and Discussion 3.1 LC-MS of Drug Product Lots The individual samples of the various DP lots were subjected to intact LC-MS analyses on a RP C4 column using a UPLC interfaced to the Waters LCT Premiere XE TOF MS. The UV profile of the region of the RP Lot K chromatogram, along with the expanded 24 - 25.7 min. region of the UV–TIC chromatogram, is shown in Fig. 2. Following MaxEnt1 processing and deconvolution of the 24.5 – 25.3 min. region of the TIC chromatogram (containing both major and minor peaks), the spectrum in Fig. 3 was obtained. The parent, unmodified species (calculated average MW 35054.15) is designated as ―M‖ in this and all subsequent spectra for the DP and DS lots. A glycated variant (+ 1 Glc) at +162 mu is clearly seen, along with an oxidized form (+ Ox) and ACN and TFA adducts. Extracted ion chromatograms were initially obtained for the native and glycated variants of DP Lot K spectra, but these showed unresolved, overlapping chromatographic profiles indicating poor resolution of these species (data not shown). Instead, a different strategy was chosen whereby 3 separate, consecutive time windows within the TIC chromatogram of Fig. 2B were processed and deconvoluted separately by the MaxEnt1 method. The TIC showing the separate time windows is shown in Fig. 4. The processed spectra for the 3 chromatographic windows are given in Fig. 5 A-C.

Fig 5A clearly shows the mono- and di-oxidized variants of DP Lot K, which are found in the smaller, early eluting peak in Fig 4. The unmodified version is largely absent from this spectrum. Processing of the following elution time window (24.7 – 24.89 min) gave the spectrum in Fig 5B. The spectrum in Fig. 5B shows the front part of the Lot K main peak to contain significant amounts of the glycated variant, in addition to the unmodified parent ion, and TFA and ACN adducts. The oxidized derivative is largely absent. When the trailing half of the main peak (Fig 4 (green)) is MaxEnt1 processed, the spectrum in Fig 5C is obtained. The later eluting, trailing half of the main peak in Fig. 4 contains primarily the unmodified DP and its TFA/ACN adducts, but no evidence of the glycated variant seen in Fig 5B. Thus, there is overlap in the retention behaviour of glycated and unmodified components of Erwinase DP under these chromatographic conditions, which is revealed following separate deconvolution of consecutive time windows of the TIC. Attempts to separate glycated from unmodified DP by gradient adjustments were unsuccessful. The MaxEnt1 deconvoluted, centroided spectrum of the combined TIC windows and corresponding peak list are given in Fig. 6 and Table 2, respectively.

Peak No.

Calculated Observed Intensity Mass Mass (Counts) 8

% Total

Mass error (ppm)

Possible Modifications

1 2 3 4 5 6

35037.266 35054.152 35070.148 35082.148 35095.180 35130.066

35038.816 35055.652 35071.996 35082.750 35096.789 35130.359

1806 15000 5100 2993 7381 1837

3.09 25.69 8.74 5.13 12.64 3.15

44.2 42.8 52.7 17.2 45.8 8.3

7

35155.223

35156.156

1094

1.87

26.5

8 9 10 11

35168.145 35184.141 35196.141 35216.207

35169.949 35185.469 35195.742 35217.035

3369 1254 1468 3261

5.77 2.15 2.51 5.59

51.3 37.7 -11.3 23.5

12

35227.176

35227.695

530

0.91

14.7

13 14

35260.172 35280.188

35259.234 35281.266

785 397

1.34 0.68

-26.6 30.6

15

35300.227

35300.352

481

0.82

3.5

16

35317.219

35316.762

314

0.54

-12.9

17

35419.965

35419.215

61

0.11

-21.2

Dehydration S(1) Unmodified Oxidation Formyl-N-term. + ACN + K (2) +Guanidine (2), Deamidation Succinimide, N(1) + TFA Oxidation, +TFA Formyl-N-term., +TFA + 1 Glc Deamidation N (1), +1 Glc, Formyl K (1), Dehydration S(1) +Glc, +Na (2) + Glc, Oxidation x 2 M (2) + Glc, Deamidation N (2), + ACN (2) +Glc, Oxidation x 2, Formyl-N-term., +ACN Oxidation x2 M (2), Deamidation N (2),Glycation (1),Oxidation M(2),+Fe3(2)

Table 2. MaxEnt1 Lot K peak list derived from Fig. 6

The MaxEnt 1 processing method revealed DP variants with masses consistent with modifications listed in the right column, based on predicted mass differences. These assignments were done manually, by comparison with centroided spectral results obtained by BiopharmaLynx processing (Table 3). A number of Erwinase-related peaks above the mass of 35,217 seen in the spectrum in Fig. 6 were not listed in Table 2, which is why the ―% Total‖ column does not add up to 100.Their observed mass differences from the native species could not be assigned to known modifications, either because the mass changes found do not correlate with those calculated for known covalent changes/salt adducts found in databases, or the mass accuracy errors were not within the windows of confidence (± 55ppm) selected for assigning specific modifiers. The total glycation level which could be assigned following MaxEnt1 processing was about 10.0% Further processing by the BiopharmaLynx™ method identified and quantified many of the derivatives in Table 2, plus others, including a doubly glycated species (1.57%). The results are given in Table 3. Table 3. BiopharmaLynx-processed Lot K TIC chromatogram

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Modifiers Unmodified Lot K + ACN Oxidation M(1) +TFA(1) Formyl N-TERM (1) Deamidation Succinimide N(1) +Guanidine (2), Deamidation Succinimide N(1) Glycation (1) +Acetonitrile (1), Oxidation M(1) +K(2) Oxidation M(1), +TFA(1) +Na(2),Glycation (1) Deamidation N(2),Glycation (1),+Acetonitrile(2) Formyl N-TERM(1),+TFA(1) Oxidation x2 M(2),Deamidation N(2),Glycation(2),Oxidation M(1) Glycation(1),+TFA(2) Glycation(1),Oxidation M(1),Deamidation Succinimide N(1),Dehydration S(1),+TFA(3) Oxidation x2 M(1),Deamidation N(1),Glycation(1),Formyl NTERM(1),+Fe3(2),Dehydration S(1) Oxidation x2 M(2),Deamidation N(2),Glycation(1),Oxidation M(2),+Fe3(2)

Calculated Mass (Da)

Observed Mass (Da)

Intensity (Counts)

Intensity (%Total)

35054.1523 35095.1797 35070.1484 35168.1445 35082.1484 35037.1289 35155.2227 35216.207 35111.1758 35130.0664 35184.1406 35260.1719 35300.2266 35196.1406

35055.625 35096.5195 35070.7305 35169.8984 35082.8164 35038.2422 35155.9219 35217.625 35111.2383 35131.1328 35184.7266 35259.4883 35301.4375 35196.043

166362.1 60247.9 49301.4 35880.2 32424.8 30763 21034.4 20505.4 20237.8 13929.8 11982.7 10955.9 10499.6 9462.9

31.97 11.58 9.48 6.90 6.23 5.91 4.04 3.94 3.89 2.68 2.30 2.11 2.02 1.82

Mass Error (ppm) 42 38.2 16.6 49.9 19 31.8 19.9 40.3 1.8 30.4 16.7 -19.4 34.3 -2.8

35460.2031

35461.625

8165.7

1.57

40.1

35444.1914

35445.2812

5224.5

1.00

30.7

35539.1445

35540.6914

4747

0.91

43.5

35364.9883

35366.418

4552.4

0.87

40.4

35419.9648

35420.1875

4041.9

0.78

6.3

The BiopharmaLynx™ processing method applies a refinement of the MaxEnt1 deconvolution method, based partly on Lock Mass correction and other factors, to improve mass accuracy. Total glycation level was found to be 13.2%. The increased levels of glycation seen using BiopharmaLynx processing(13.2 vs 10%) is at least partly attributable to its identification of additional glycated variants compared to the MaxEnt1-processed data. This observation that over 13% of DP Lot K is glycated suggests that Erwinase glycation readily occurs to an appreciable extent either during the formulation-fill-finish processing step, the following 3 month period as a lyophilised powder, or both (DP Lot K was released in Aug. 2016 and this analysis is from Nov. 2016). We assessed the glycation levels of older lots of Erwinase DP by LC-MS, as evidence for continued glycation occurring in the freezedried state under nominal storage (+2 – 8oC) conditions. In Fig. 7, the overlapping RP UV profiles of DP Lots C and G are shown. The UV profiles are similar to that of DP Lot K (Fig. 2B), except that for DP Lot G, the minor peak at 32.8 – 33.0 min., oxidized has increased resolution from the main peak (Fig. 7), whereas this peak co-elutes as a front shoulder on the main peak for DP Lot C. This is largely due to the broadening of the main peak in the older DP Lot C, due to co-eluting glycated variants as for DP Lot K (above). Following MaxEnt1 processing of the 32.7 – 33.3 min. region of the corresponding TIC chromatogram for DP Lot G and deconvolution, the spectrum in Fig. 8 was obtained. The main peak in Fig. 8 is the unmodified Erwinase (M), with ACN and TFA adducts also identified. The mono-glycated (+1 Glc) and di-glycated (+2 Glc) variants are clearly observed, and further processing by BiopharmaLynx identified and quantified additional levels of dehydration, deamidated variants, and salt adducts in addition to the above. Total glycation was found to be 23.9%. DP Lot G was released in January 2015, more than 1.5 years prior to DP Lot K but was still within its 3 year shelf life when tested. The observation that glycation levels nearly doubled over this 1.5 year period relative to DP Lot K levels suggests this modification

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continues within the freeze-dried powder at low temperature over time, as opposed to solely in the formulation-fill-finish processing step. The TIC chromatogram corresponding to the UV profile of DP Lot C in Fig. 7 was also MaxEnt1 deconvoluted and processed by BiopharmaLynx in the same manner as for DP Lot G. Total glycation for Lot C is at 41.5%, which also includes a >2% level of tri-glycation (Table 4). DP Lot C was released in March, 2010, and was 6.5 years old at testing. It was also stored at low (+2 – 8oC) temperature during this period. In DP Lot C, there is also no resolution between the UV minor (oxidized) and major peak as seen for Lot G (Fig. 7). As mentioned above, this suggests that the accumulation of glycated derivatives co-eluting with the Erwinase main peak prevent resolution from the oxidized variants.

Table 4 DP Lot C D (+2 – 8oC) E F G I K

Glycation summaries for Erwinase DP Lots Release Date % Mono-Glycated Mar 2010 Mar 2012 Nov 2012 Aug 2014 Jan 2015 Nov 2015 Aug 2016

26.6 26.6 26.9 20.0 19.3 19.5 11.7

% Di-Glycated 12.5 9.0 4.1 2.9 4.6 <2 <2

% Total Glycation 41.51 35.6 31.0 22.9 23.9 19.5 13.2

1

also includes tri-glycated species

Other Erwinase DP lots were subjected to intact LC-MS analyses as above to quantitate glycation levels and results for all DP lots tested are given in Table 4 and the plot in Fig. 9. The glycation levels (Fig. 9, right column) seen for each Erwinase DP lot generally increase with age. If the trend line in Fig 9 is extrapolated to 0 months, it indicates that glycation has already occurred to a significant level (~10%) during the formulation/fill/finish step prior to completion of lyophilisation. This process can take as long as 2 days. This observation requires further study to identify how glycation can be minimised at this early stage. It should be noted here that glycation is only being measured as the formation of the stable Amadori ketoamine derivative (+162). Further reaction products are not identified by the BiopharmaLynx processing method, since the structures for such intermediates have not been determined, and thus are not included in the program‘s modifications database. It is known that the Amadori product can undergo crosslinking, dehydration, or further oxidative reactions leading to carboxymethyllysine, pentosidine, or pyrraline derivatives, etc. in other matrices. Identification and quantitation of these degradants may allow a more accurate comparison of the overall glycation extent for the different DP lots. Although absolute quantitation of glycation levels by MS may be affected somewhat by differences in ionisation efficiencies between glycated derivatives and native species, any such variations should be consistent across the range of DP lots tested. This allows for the relative comparison of glycation levels of different DP lots with age. 3.2 LC-MS of Erwinase Drug Substance (DS) Lots As mentioned above, the fermentation and downstream processing of Erwinase are unlikely sources of glycation, since the composition of the yeast extract used as a nutrient source in the fermentation of the Erwinia chrysanthemi (source of Erwinase) is reported by the manufacturer to be devoid of mono- and disaccharides. We examined the LC-MS results

11

of 2 Erwinase DS lots (R and S), acquired before formulation with excipient to yield the corresponding DP lots, for evidence of glycation to confirm this. DS Lot S was formulated with glucose excipient to give DP Lot G (Section 3.1 above) while DS Lot R was formulated with glucose to yield DP lots L - O. The TIC profile of DS Lot R is similar to that of the DP lots above. The MaxEnt1 processed spectrum of the 33.1 – 33.7 min. region of the TIC chromatogram is given in Fig. 10.

This spectrum, while clearly showing the unmodified and oxidized Erwinase and salt/solvent adducts, provides no evidence of glycated variants in the relevant mass region. This would be expected if this DS lot had not been exposed to reducing sugars. A sample of DS Lot S was also analysed by LC-MS in a similar manner, and the MaxEnt1-processed spectrum of the TIC chromatogram was obtained. As in the DS Lot R spectrum, there is no evidence of glycated derivatives in this sample. These analyses support the absence of observable glycation events both during the fermentation and the downstream purification processing in Drug Substance manufacturing.

3.3 DP Lot D Stability Samples As demonstrated above, DP glycation continues within the lyophilised state at +2 – 8oC. For the purpose of evaluating the impact of storage temperature on DP stability, DP Lot D was monitored in its lyophilised state within the Erwinase stability program at 3 different temperatures (-80oC, -20oC, and +2-8oC) under controlled conditions. It was originally released in March, 2012. We were able to test a sample of each at the 4 year, 11 month time point by RP-UPLC-MS analysis as in Section 3.1 above. Each of the 3 stability samples were analysed in the same manner as the DP lots above (see Material and Methods). The MaxEnt1 results for the DP Lot D sample held at -80oC are given in Fig. 11. A mono-glycated Erwinase variant is clearly identified in the spectrum in Fig. 11 and in the BiopharmaLynx-processed data. The glycated variants comprise 17.1% of the total Erwinase species. Of interest is the fact that the glycation level of the -80oC (17.1%) sample, even at 4 years, 11 months after release, is comparable to those of DP Lot I (19.5%) and Lot K (13.2%) (Table 4). These were only 12 and 3 months old, respectively, at time of analysis. The big differences, obviously, are the temperatures at which the 2 sets of samples were stored, with DP Lots I and K held at nominal conditions (+2 - 8oC) before testing vs DP Lot D which was held at -80oC. The -20oC and +2 - 8oC stability samples of DP Lot D were also subjected to LC-MS analysis in a similar manner, and the UV chromatograms of each were overlaid with that of the -80oC Lot D sample and shown in Fig. 12. From Fig. 12 above, it is seen that the loss of resolution of the minor (oxidized) and major (main) peaks for the +2-8oC DP Lot D sample, compared to the 2 others, is accompanied by a significant increase in main peak broadening. This resembles the differences in main peak broadening between DP Lot G and DP Lot C (Fig. 7), which were due to differences in glycation levels, as the glycated variants of DP did not resolve from the unmodified DP under these conditions. The overlapping retention behaviour of glycated and non-glycated variants can be seen when the TIC chromatogram for Lot D (+2-8oC, Fig. 13) is processed in consecutive time windows as for DP Lot K (Fig 4) above. The TIC chromatogram for DP Lot D (+2-8oC ) is shown in Fig 13, with the individual, coloured RT windows processed separately by MaxEnt1. The processed spectra for the 3 chromatographic windows are given in Fig. 14 A-C.

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It is clear from Fig 14A that the leading edge of the TIC in Fig. 13 comprises a complex mixture of mostly oxidized and glycated variants of Lot D, with very little of the unmodified Lot D seen. This is consistent with the observations of Lot K TIC early region, where mainly the oxidized variant was seen (Fig 5A). The 33.8 – 34 min region from Fig. 13 was also MaxEnt1-processed separately, with the spectrum given in Fig 14B. The spectrum in Fig 14B is dominated by the presence of glycated derivatives of Lot D, with the unmodified Erwinase being a minor component of this mixture. This again illustrates the overlapping nature of the elution profiles of the glycated and native Erwinase variants in RP chromatography, even though the gradient in this case was extended in an attempt to get better resolution of native and glycated species. Finally, the last RT window (green) of Lot D TIC main peak (34-34.4 min) in Fig. 13 was processed by MaxEnt1 to give the spectrum in Fig 14C. Fig 14C shows the glycated variant to be a minor component of this part of the TIC, consistent with the results obtained for the latter- eluting component (Fig. 5C) of DP Lot K, where the glycated derivative was largely absent. The TIC chromatograms from DP Lot D (-20oC) and Lot D(-80oC) were deconvoluted and processed by BiopharmaLynx as for the Lot D (+2 – 8o C ) stability sample. The glycation levels seen in all 3 samples are summarized below in Table 5:

Table 5 Glycation summaries for Erwinase DP Lot D, 4yr, 11 month stability time point, stored at the indicated temperature conditions

Temperature

% monoglycation

% diglycation

% total glycation

-80o C

17.1

<1

17.1

-20o C

12.1

<1

12.1

+2o – 8o C

26.6

9.0

35.6

A dramatic increase in the levels of DP Lot D glycation is seen (35.6% of total) when it is stored at +2 – 8oC vs Lot D samples kept at -20oC (12.1%) or -80oC (17.1%). The +2 - 8oC Lot D sample also contains di-glycated species, while the differences in glycation levels between the -20oC vs -80oC samples are minimal (Table 5). The slight increase in glycation seen for the -80oC vs. the -20oC stability samples may be due to other storage condition

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differences such as moisture content or light exposure. Overall, these results indicate that these 2 low temperature conditions are more effective at preventing Erwinase glycation in the lyophilised state. These observations suggest the likelihood that differences in glycation levels can be expected between DP lots stored at +2 – 8oC vs -20oC (or -80oC) within their normal shelf life. 3.5 Capillary isoelectric focussing Capillary isoelectric focussing (cIEF), as an orthogonal separation technique to reversedphase chromatography, was also used to monitor glycation of Erwinase DS (EDS) and DP lots. Focussing first on Erwinase DP lots, the following samples were analysed by cIEF: DP Lots A, B, and H. DP Lot A, released in 1990, was found by LC-MS analysis to be heavily glycated (> 70%), including tri and tetra-glycated species (data not shown). DP Lot B, released in 2008, was glycated to a level of 38%, while DP Lot H, at 1.5 years after release, was found to be 17% glycated by LC-MS. These 3 DP lots were compared to DS Lot R (EDS) as a reference standard. The EDS reference standard contains only one peak at a pI of 7.7 which is similar to that found in previous research done within our group (Fig. 15) (Gervais and King, 2014). When this is compared to the three DP lots described above, it is clear that there are significant differences between the DS and all three DP lots. These include the appearance of multiple resolved acidic peaks in the DP lots, as well as distinct shoulders on the fronts of the main peaks, giving rise to peak broadening (Fig. 16).

The magnitude of the broadening appears to correlate with Lot age, with the effect greatest for Lot A (318 months at testing), followed by Lot B (103 months) and Lot H (18 months). This contrasts with DS Lot R main peak, which is not glycated (Fig. 15). This suggests that glycation does not necessarily give rise to resolvable acidic peaks in these lots, but can have more subtle effects on cIEF behavior. The acidic peaks are roughly evenly spaced with around 0.2 pI units between each of them, as shown in Fig. 17.

It is hypothesised that these peaks could result from multiple glycation events. Glycation of antibodies has been shown previously to produce acidic peaks by cIEF analysis, as has deamidation (Wu and Huang, 2006); however, previous work by our group has shown that acidic peaks found in ion exchange separation of Erwinase are not due to deamidation (Gervais et al., 2015). Furthermore, the intact MS analysis shows glycation is a major component in all three DP lots. The total area of the acidic peaks seen in Fig 17 (insert) is 17.25%, which is less than the 38% levels of total glycation found by LC-MS for Lot B. It may be that single glycation events per subunit may be contained in front shoulders on the main peak, whereas resolved peaks in the acidic region may correspond to multiple glycation events per subunit, resulting in a greater impact on charge variation. Alternatively, other residues may also be glycated with different effects on net charge. These observations require further investigation to characterize effects of glycation on cIEF behaviour. The pI shift of 0.2 pI units in Fig. 17 is higher than the previously seen 0.05 pI shift associated with glycation and the 0.09 pI theoretical shift (Quan et al., 2008). This may again result from the impact of multiple glycation events. It is also well known that pI values are heavily influenced by environment. Due to the high stability of the assembled Erwinase tetramer, it must be subjected to rigorous denaturation conditions for cIEF analysis, which is not the case for the aforementioned antibody (Wu and Huang, 2006). Therefore, this more

14

rigorous treatment could explain the difference in shifts in pI values seen for glycation events. A DS batch (DS Lot R) was also tested using this method. As can be seen, there are no acidic peaks observed in the EDS electropherogram (Fig. 15). This supports the findings of the LC-MS data and strongly implies that the addition of glucose excipient at the formulation/freeze drying step is the likely source of the glycation events observed in this study. 4. Conclusions In this study, glycation of Erwinase DP has been thoroughly further characterised using mass spectrometry. We have looked at glycation levels of 7 Erwinase Drug Product lots which have been released and stored under nominal (+2o - 8oC) or lower temperature (-20o or -80oC) conditions in lyophilised form over a 7 year time window. In the LC-MS of the intact protein species, there is a general correlation of glycation levels with DP lot age (Fig. 9). We have observed the presence of glycated variants in Drug Product lots whose tryptic maps have undergone LC-MS analyses in the past, but the previous studies were limited in scope. Previously, a gel-based proteomic study using 2-dimensional gel electrophoresis of a DP lot revealed 4 spots indicating post-translational modifications, and LC-MS analyses of in-gel digests identified glycation among them, although it was not quantified (Bae et al., 2011). This report is the first we are aware of that evaluates glycation of a lyophilised biotherapeutic formulated with a reducing sugar excipient at low temperature over an extended time period. The levels of glycation observed correlate generally with age of the DP lot studied (Table 4). Importantly, glycation appears to proceed continuously with time during +2 - 8oC storage in the freeze-dried state, as evidenced by the level of DP Lot C modification seen here, where > 41% of the protein was glycated. The dramatic difference in glycation levels observed between 2 otherwise identical samples of DP Lot D, stored at +2 – 8oC vs -20oC over a nearly 5-year period (Table 5), illustrates the dependence of the kinetics of this reaction on temperature, even in this range. This was unexpected and suggests that, since lyophilised Erwinase reference standards are kept at +2 – 8oC over their lifetime, their performance in analytical assays would likely be improved if they were stored at -20oC or lower for this purpose.

Acknowledgements The authors would like to thank Roger Hinton, Managing Director of Porton Biopharma, Ltd., and Julie Miller, Director of Development, for allowing the facilities and resources to be available for this study; Stuart Smith, Head of Analytical Development, for facilitating these studies; Dave Gervais, for kindly providing a critical review of this manuscript and offering helpful suggestions, and the other members of the PBL team who kindly provided DP and DS samples for analyses. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors, and was supported entirely by Porton Biopharma, Ltd. PBL did not have any role in study design, data collection, report writing, or decision to publish this work.

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CRediT author statement Patrick Kanda: Conceptualization, Methodology, Software, Validation, Formal Analysis, Investigation, Resources, Writing – Original Draft, Visualization, Project Administration. Thomas Minshull: Methodology (capillary isoelectric focussing), Software, Formal Analysis, Investigation, Writing- Original Draft, Writing-Review & Editing, Visualisation. References Andya, J.D., Maa, Y.G., Costantino, H.R., Nguyen, P.A., Dasovich, N., Sweeney, T.D., Hsu, C.C., Shire, S.J., 1999. The effect of formulation excipients on protein stability and

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aerosol performance of spray-dried powders of a recombinant humanized anti-IgE monoclonal antibody, Pharm. Res. 16, 350 – 358. Arakawa, T., Timasheff, S.N., 1982. Stabilization of protein structure by sugars, Biochemistry 21, 6536-6544. Bae, N., Pollak, A., G. Lubec, G., 2011. Proteins from Erwinia asparaginase Erwinase® and E. coli asparaginase 2 MEDAC® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear, Electrophoresis 32, 1824 – 1828. Bozhinov, A.S., Boyanova, M., Niwa, T., Ivanov, I., Mironova, R., 2010. Evidence for the presence of glycation adducts in protein therapeutics. Biotechnol. & Biotechnol. Equip. 24, 1904-1909. Carpenter, J.F., Crowe, L.M., Crowe, J.H., 1987. Stabilization of phosphofructokinase with sugars during freeze-drying: characterisation of enhanced protection in the presence of divalent cations, Biochim. Biophys. Acta 923, 109-115. Fischer, S., Hoernschemeyer, J., Mahler, H.C., 2008. Glycation during storage and administration of monoclonal antibody formulations, Eur. J. Pharm. Biopharm. 70, 42-50. Gadgil, H.S., Bondarenko, P.V., Treuheit, M.J., Ren, D., 2007. Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method, Anal. Chem. 79, 5991-5999. Gervais, D., King, D., 2014. Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea–urea mixtures, Anal. Biochem. 465, 90-95. Gervais, D., King, D., Kanda, P., Foote, N., Elliott, L., Brown, P., Lee, N.O., Thalassinos, K., Pizzey, C., Rambo, R., Minshull, T.C., Dickman, M.J., Smith, S., 2015. Structural characterisation of non-deamidated acidic variants of Erwinia chrysanthemi L-asparaginase using Small-Angle X-ray scattering and Ion-Mobility Mass Spectrometry, Pharm. Res. 32, 3636-3648. Hellman, K., Miller, D.S., Cammack, K.A., 1983. The effect of freeze-drying on the quaternary structure of L-asparaginase from Erwinia carotovora, Biochim. Biophys. Acta 749, 133-142. Kassaar, O., Pereira Morais, M., Xu, S., Adam, E.L., Chamberlain, R.C., Jenkins, B., James, T.D., Francis, P.T., Ward, S., Williams, R.J., van den Elsen, J., 2017. Macrophage Migration Inhibitory Factor is subjected to glucose modification and oxidation in Alzheimer‘s Disease, Sci. Rep. 7: 42874, 1-11. DOI: 10.1038/srep42874. Kennedy, D.M., Skillen, A.W., Self, C.H., 1994. Glycation of monoclonal antibodies impairs their ability to bind antigen, Clin. Exp. Immunology 98, 245–251. Leblanc, Y., Bihoreau, N., Jube, M., Andre, M.-H., Tellier, Z., Chevreux, G., 2016. Glycation of polyclonal IgGs: Effect of sugar excipients during stability studies, Eur. J. Pharm. Biopharm. 102, 185 – 190. Luthra, M., Balasubramanian, D., 1993. Nonenzymatic glycation alters protein structure and stability. A study of two eye lens crystallins, J. Biol. Chem. 268, 18119 – 18127.

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Maillard, L.C., 1912. Action of amino acids on sugars. Formation of melanoidins in a methodical way, Compt. Rend. 154, 66-68. Quan, C.P., Wu, S., Dasovich, N., Hsu, C., Patapoff, T., Canova-Davis, E., 1999. Susceptibility of rhDNase I to glycation in the dry-powder state, Anal. Chem. 71, 4445 4454. Quan, C., Alcala, E., Petkovska, I., Matthews, D., Canova-Davis, E., Taticek, R., Ma, S., 2008. A study in glycation of a therapeutic recombinant humanized monoclonal antibody: Where it is, how it got there, and how it affects charge-based behaviour, Anal. Biochem. 373, 179-191. TreDenick, T., 2008. Process Development Report: Development of the Depyrogenation, Formulation, Filling and Lyophilisation Process for Erwinase®, Document Number BTLHPA-DR-005, 61. Ulrich, P., Cerami, A., 2001. Protein glycation, diabetes, and aging. Recent Progress Horm. Res. 56, 1-21. Vrdoljak, A., Trescec, A., Benko, B., Hecimovic, D., Simic, M., 2004. In vitro glycation of human immunoglobulin G, Clinica Chimica Acta 345,105 – 111. Ward, K.R., Adams, G.D.J., Alpar, H.O., Irwin, W.J., 1999. Protection of the enzyme l asparaginase during lyophilisation—a molecular modelling approach to predict required level of lyoprotectant, Int. J. Pharmaceutics 187, 153 – 162. Wu, J., Huang, T., 2006. Peak identification in capillary isoelectric focusing using the concept of relative peak position as determined by two isoelectric point markers, Electrophoresis 27, 3584–3590. Yang, Y., Primack, R., Frohn, M., Wang, W., Luan, P., Retter, M.W., Flynn, G.C., 2014. Impact of glycation on antibody clearance. AAPS Journal 17, 237-244. Zhang, Q., Ames, J.M., Smith, R.D., Baynes, J.W., Metz, T.O., 2009. A perspective on the Maillard reaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease, J. Proteome Res. 8, 754 – 769. Zheng, X., Wu, S.L., Hancock, W.S., 2006. Glycation of interferon-beta-1b and human serum albumin in a lyophilised glucose formulation Part III: Application of proteomic analysis to the manufacture of biologic drugs, Int. J. Pharmaceutics 322, 136-145.

18

FIG. 1

19

FIG.2

20

FIG.3

21

FI.G4

22

FIG.5

23

24

FIG 6.

25

FIG. 7

26

FIG. 8

27

FIG.9

28

FIG. 10

29

FIG. 11

30

FIG. 12

31

FIG.13

32

FIG. 14

33

FIG. 15

FIG.16

34

35

FIG.17

36

Figure legends Fig. 1. Formation of the Amadori product following reaction of the open-chain form of glucose with the lysine -amino group of a protein (reprinted with permission from Quan et al., 2008) Fig. 2. (A) UV chromatogram of DP Lot K, (B) Expanded 24 – 25.7 min window of the stacked UV and TIC plots of DP Lot K. Fig. 3. MaxEnt1 deconvoluted spectrum of DP Lot K showing the mono-glycated and oxidized derivatives, along with TFA and ACN adducts. Fig. 4. Lot K TIC chromatogram 24.2 – 25.4 min. region, shown as 3 consecutive (yellow, red, green) elution time windows. Fig. 5A. MaxEnt1 deconvoluted 24.3 – 24.7 min window (yellow) of the Lot K TIC chromatogram in Fig. 4. Fig. 5B. MaxEnt1 deconvoluted 24.7 – 24.89 min window (red) of the Lot K TIC chromatogram in Fig. 4, showing the presence of the glycated (+Glc) derivative Fig. 5C. MaxEnt1 deconvoluted 24.89 – 25.2 min window (green) of the Lot K TIC chromatogram in Fig. 4, showing the absence of the glycated derivative. Fig. 6. MaxEnt 1 centroided spectrum of Lot K obtained by deconvolution of the 24.3 – 25.2 min region of the TIC in Fig. 4. Fig. 7. Overlaid UV chromatograms of DP Lots C (black) and G (blue) in the 31.8 – 34.0 min window Fig. 8. MaxEnt1 deconvoluted spectrum of DP Lot G showing mono- and diglycated derivatives, along with TFA and ACN adducts. Fig. 9. Plot of DP glycation levels vs. lot age, using the data from Table 4. The dotted line denotes the glycation trend with age. Fig. 10. MaxEnt1 deconvoluted spectrum of DS Lot R showing the oxidized product, the ACN and TFA adducts, and the absence of glycated derivatives. Fig. 11. MaxEnt1 deconvoluted spectrum of Lot D -80oC showing the ACN and TFA adducts along with the mono-glycated derivative. Fig. 12. Overlaid tracings of the UV chromatograms of DP Lot D stored at the temperatures indicated. The tracings for the -20oC (red) and -80oC (blue) samples show significant resolution of the minor, oxidized peak from the larger main peak. However, the +2 - 8oC sample (black) tracing shows almost no resolution of these 2 peaks. Fig. 13. Lot D (+2-8oC) TIC chromatogram 33.2 – 34.5 min. region shown as 3 consecutive (yellow, red, green) elution time windows. Fig 14A. MaxEnt1 deconvoluted 33.2 – 33.8 min window (yellow) of the Lot D TIC chromatogram in Fig. 13, showing primarily oxidized and glycated derivatives. Fig 14B. MaxEnt1 deconvoluted 33.8 – 34 min window (red) of the Lot D TIC chromatogram in Fig. 13, showing the presence of the glycated (+1 Glc and +2 Glc) derivatives. Fig 14C. MaxEnt1 deconvoluted 34 – 34.4 min window (green) of the Lot D TIC chromatogram in Fig. 13, showing the presence of the unmodified and glycated derivatives. Fig. 15. cIEF electropherogram of Erwinase Drug Substance Lot R (EDS). The unlabelled peaks at 4.6 and 8.4 pI are pI markers. Fig. 16. A) cIEF electropherogram of Erwinase DS Lot R and three DP lots of different ages in a stack overlay. In each of the DP batches, additional peaks can be seen in the acidic region, along with a small shift in the pI of the main peak. B) an expanded section of DP Lot H, highlighting the shoulder detail of the main peak. 37

Fig. 17. A cIEF electropherogram of DP Lot B, exemplifying the acidic peaks within this sample, to the left of the Erwinase Drug Product (EDP) main peak. There is roughly a delta of 0.2 pI units between these acidic peaks. The peak areas and % areas are given in the insert.

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Graphic abstract

39