Assessing the impact of formulation on protein-based biotherapeutics

Bradley, Derek1; Stocca, Alessia2; Burke, Mary1; Roche, James J.2

1 BioClin Research Laboratories, IDA Technology Park, Garrycastle, Athlone, Co. Westmeath, Ireland

2 Biosciences Research Institute, Athlone Institute of Technology, Dublin Road, Athlone, Co Westmeath, Ireland.

AbstractBiopharmaceuticals are a class of revolutionary drugs that have been developed to treat a diverse range of serious and often life-threatening diseases, such as cancer. As with small molecule drugs, they must be tested to ensure they meet predefined specifications prior to release. However, owing to their complexity, the analytical challenges for biopharmaceuticals are far greater than for small molecule drugs. Being protein in nature, biopharmaceutical structure can be dramatically altered by formulation excipients. This may lead to an inefficacious drug, or indeed, one which may have a significantly altered safety profile.

In this research, the following range of analytical techniques have been applied to two assumed-identical asparaginase formulations, a biopharmaceutical that is used to treat acute lymphoblastic leukaemia (ALL) in children:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Intact mass (‘top-down’) analysis – mass spectrometry

Peptide mass fingerprinting (primary structure analysis)

Amino acid compositional analysis

Spectroscopic analysis – circular dichroism (CD)

Results show that the formulations exhibit highly similar responses in some analytical techniques, but not others. This demonstrates that in order to characterise the impact of formulation on biological medicines, an orthogonal analytical approach, comprising a battery of techniques which offer complimentary information, should be employed.

IntroductionBiopharmaceuticals are protein-based therapeutics, which only function as intended when they adopt the correct structure and three-dimensional shape. Proteins have several layers of assembly, each of which play a critical role in determining overall protein shape. Alterations at any level of structure may result in an incorrectly folded protein, which could give rise to a molecule with reduced efficacy, or potentially trigger a harmful immune response. (1-2)

Formulation of a biopharmaceutical product can dramatically influence the stability and structure of the protein, due to interactions between the amino acid variable groups and formulation excipients or processes. (3) For example, development of modified forms (such as aggregates) may be promoted during lyophilisation, (4) while certain excipients may contribute to chemical modifications, such as oxidation.(5) Therefore, a key aspect of product development is that the influence of formulation on stability and structure of biologics should be thoroughly characterised.

This research is a joint industry/academia collaborative effort which aims to explore and develop methods to characterise biopharmaceuticals. Development of robust techniques capable of reliably detecting differences in biopharmaceutical structure is critical to ensuring patient safety. The rapid emergence of biosimilars (copy-versions

of innovator biologics authorised under abbreviated licensure pathways) underlines the increasing need for availability of such techniques.

Methods and MaterialsSamples for all tests were lyophilisates which were reconstituted in water according to manufacturer’s instructions – final concentration was approximately 13.0 mg/mL. Reconstituted material was stored frozen at -80ºC in single-use aliquots. When required, aliquots were thawed at room temperature. The following sections briefly describe the methods for each test (experimental settings were optimised for each test during pilot-scale experiments) – further details can be obtained by contacting the author.

SDS-PAGE: Samples were diluted with water to 1.0, 0.5 and 0.2 mg/mL. 100 µL of each dilution was combined with an equal volume of 2X Laemmli sample buffer containing 100 mM DTT – this gives final protein concentrations of 0.5, 0.25 and 0.1 mg/mL, respectively. 20 µL of each preparation was pipetted into the wells of an ‘Any-kD, Stain-free’ polyacrylamide gel (BioRad, product code # 456-8123), according to Figure 1 – this volume corresponds to a total protein load of 10, 5 and 2 µg, respectively. Samples were electrophoresed at 200 V (constant voltage) using the Mini-Protean electrophoresis system from BioRad – run time was 30 minutes. The gel was then imaged using the ‘Stain-free’-enabled ChemiDoc MP Imaging system from BioRad. Data was processed using ImageLab Software version 5.4.1.

Intact mass analysis: Samples were diluted in 50% (v/v) acetonitrile containing 0.1% (v/v) formic acid to a final concentration of 50 µg/mL. Samples were then infused using a Harvard 22 apparatus and syringe with a flow rate of 20 µL/min into the electrospray ionisation source of the API3000 mass spectrometer (ABSciex). The mass spectrometer ionisation and detection settings were as detailed in Table 1, below. Data was analysed using Analyst version 1.4 software.

Table 1: Instrument settings for intact mass analysis via MS

Detector settings Ionisation settings

Parameter Value Parameter Value

MCA 20 scans Nebuliser gas 8.00

Scan mode Profile Curtain gas 8.00

Scan type Q1 MS (Q1) Ion spray voltage 5000.00

Polarity Positive Temperature 0.00

Ion source Turbo Spray Declustering potential 80.00

MCA 20 scans Focussing potential 400.00

Step size 0.10 a.m.u. Entrance potential 10.00

Peptide mass fingerprinting: Samples were diluted to 1.0 mg/mL with water. 50 µL of each sample (corresponding to 50 µg of protein) was desalted using a solid phase extraction cartridge containing C8 resin. Samples were evaporated to dryness in a vacuum centrifuge, then resuspended in 20 µL of 50 mM ammonium bicarbonate containing 8 M urea. 5 µL of 45 mM dithiothreitol (DTT) was added followed by incubation at 60ºC for 45 minutes, during which disulfide bonds were reduced. Then, 10 µL of iodoacetamide (IAA) was added, and tubes incubated at room temperature in the dark for 45 minutes, during which free cysteines were alkylated to prevent disulfide bonds reforming. A further 5 µL of 45 mM DTT was then added to neutralise excess IAA. Final volume was brought to 200 µL with water in order to dilute urea to below 1.0 M, as high urea concentration is not compatible with trypsin. 2 µL of 1.0 mg/mL sequencing-grade trypsin was then added, and tubes incubated at 37ºC for 24 hours. Following incubation, digestion was stopped by freezing the samples at -20ºC until required for analysis. Samples were diluted

1:1 with mobile phase A (see below) and 40 µL injected. Analysis was on the API3000 LC-MS/MS (ABSciex) system with Perkin Elmer 200 Series autosampler and quaternary pump. Mass spectrometer settings were as detailed in Table 1, with the exception that source temperature was 400ºC. The column was a C18-AR type, with dimensions of 4.0 mm (I.D.) X 150 mm (length) with 3 µm particle size. Samples were eluted with a 120 minute linear gradient from 2% (v/v) acetonitrile with 0.1% (v/v) formic (Mobile phase A) acid to 50% (v/v) acetonitrile with 0.1% (v/v) formic acid (Mobile phase B). Flow rate was 1.0 mL/min, which was split with approximately 0.5 mL/min going to the source (the remainder going to waste). Data was collected and analysed using Analyst version 1.4 software.

Amino acid compositional analysis: Samples were prepared at a concentration of 1.0 mg/mL. 2 µL of each sample (2 µg of protein) was added to an autosampler vial and evaporated to dryness. 200 µL of 6 N HCl containing 0.1% (w/v) phenol was added, then the headspace of tubes purged with a stream of nitrogen before being capped tightly. Tubes were then placed in a vacuum oven at 115ºC for 24 hours, during which peptide bonds are hydrolysed, releasing the free amino acids. Following hydrolysis, HCl was evaporated off, then amino acids derivatised using the Waters AccQ-Tag chemistry package. Analysis was on the Varian ProStar Fluorescence HPLC System. The column was a Nova-Pak C18, (4.0 mm I.D., 150 mm in length, 4 µm particle size). A linear gradient from 2% to 50% B over 60 minutes, with a flow rate of 1.0 mL/min was used for analysis. Injection volume was 5 µL. Standards from 0 to 100 µM of each amino acid were also prepared from commercially available amino acid standards. These were also derivatised and analysed in order to quantify the amount of each respective amino acid present in the test samples. Data collection and processing was performed using Varian Star Chromatography Workstation software.

Circular dichroism: CD analysis was performed at Applied Photophysics, Leatherhead, UK. Samples were diluted approximately 1:20 with water to reduce absorbance to about 1.8 A.U., which is required for CD analysis. A lyophilised vial of asparaginase was also analysed – this was also diluted with water in order to obtain an absorbance of approximately 1.8 A.U. Samples were scanned on the Chirascan Plus qCD Spectrometer. Analysis was performed in isothermal mode (at 20ºC) in the far UV region (260 to 180 nm), with a wavelength increment of 1 nm, and a bandwidth of 1 nm. Samples were also analysed with temperature ramping in order to compare melting temperatures. For temperature ramping experiments, the scan range was from 250 to 190 nm, wavelength increment of 1 nm, bandwidth of 1 nm, and temperature ramped from 20ºC to 90ºC in 1ºC increments. Applied Photophysics’ Global 3 software was used for data analysis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Figure 1: Left – Gel imaged following electrophoresis with bands for standards and samples

auto-detected in ImageLab software v. 5.4.1. Right – green filter applied to image revealing

the presence of additional high molecular weight protein bands (indicated by red arrows).

Table 2: Molecular masses estimated from SDS-PAGE

Lane # Sample details Molecular mass - kDa

1 Mw ladder N/A

2 R040614 – 0.1 mg/mL 33.6

3 M030614 – 0.1 mg/mL 34.0

4 R040614 – 0.25 mg/mL 33.8

5 M030614 – 0.25c mg/mL 34.2

6 R040614 – 0.5 mg/mL 33.4

7 M030614 – 0.5 mg/mL 33.0

8 Mw ladder N/A

9 Blank N/A

10 Empty N/A

Intact mass analysis via mass spectrometry

Figure 2: Centroided spectra from electrospray ionisation mass spectrometry of asparaginase samples: Left – M030614; Right – R030614. Inset – example of profile spectra before ‘centroiding’

Figure 3: Above – characteristic electrospray ionisation charge envelope for M030614 in profile; Right – calculation of intact molecular mass from charge envelope data.

Peptide mass fingerprinting via ESI LC-MS

Figure 4: Top – recombinant asparaginase; Bottom – non-recombinant asparaginase. In each Figure, the top panel represents the total ion current (TIC) for peptides. The three lower panels are extracted ion chromatograms for three selected peaks, which gives the molecular mass of the peptide fragments, which allows their identity to be confirmed.

Amino acid compositional analysis

Figure 3: Above – characteristic electrospray ionisation charge envelope for M030614 in profile; Right – calculation of intact molecular mass from charge envelope data.

Peptide mass fingerprinting via ESI LC-MS

Figure 4: Top – recombinant asparaginase; Bottom – non-recombinant asparaginase. In each Figure, the top panel represents the total ion current (TIC) for peptides. The three lower panels are extracted ion chromatograms for three selected peaks, which gives the molecular mass of the peptide fragments, which allows their identity to be confirmed.

Amino acid compositional analysis

Figure 5: Left – example standard curve showing excellent correlation; right – representative chromatogram for 75 µM derivatised standard

Table 3: Amino acid composition of asparaginase

Amino acid R040314 M170414 Mean St. Dev. %RSD

Ala 123.2 107.8 115.5 11.0 9.5

Arg 53.2 108.4 80.8 39.1 48.4

Asp 191.8 173.7 182.7 12.8 7.0

Cys 39.1 39.1 39.1 0.0 0.0

Glu 81.9 76.1 79.0 4.1 5.2

Gly 126.2 111.5 118.8 10.4 8.7

His 15.6 12.2 13.9 2.4 16.9

Ile 48.7 43.2 45.9 3.9 8.4

Leu 89.7 80.0 84.9 6.9 8.1

Lys 95.2 81.7 88.4 9.5 10.8

Met 3.6 16.1 9.9 8.8 89.1

Phe 32.1 29.0 30.6 2.2 7.1

Pro 53.0 48.1 50.6 3.4 6.8

Ser 61.2 55.3 58.3 4.1 7.1

Thr 25.2 29.6 27.4 3.1 11.4

Trp Completely destroyed during hydrolysis

Tyr 244.5 215.5 230.0 20.5 8.9

Val 40.6 40.6 40.6 0.0 0.0

Table 4: Correlation of amino acid standard curves

Amino acid r2

Lys 0.994

Ile 0.999

Leu 0.999

Asp 0.999

Ser 0.996

Glu 0.999

Gly 0.999

His 0.999

Arg 0.999

Thr 0.996

Ala 0.999

Pro 0.981

Cys 0.992

Tyr 0.979

Val 0.991

Met 0.999

Phe 0.998

Circular dichroism analysis

Figure 6: Normalised CD spectra of Non-recombinant, Recombinant, Lyophilised

Figure 7: CD spectra of Medac asparaginase during temperature ramping

Figure 8: CD spectra of recombinant asparaginase during temperature ramping

DiscussionIntact mass analysis via mass spectrometry revealed that the active ingredient in both preparations had the same molecular mass (to within 1 Da of each other, which is below the 10 p.p.m. error typically allowed when reporting intact mass results for proteins) (Figures 2 and 3). Post-translation modifications such as glycosylation, or sample degradation processes, such as oxidation or deamidation, would have a major impact on intact molecular mass. However, this was not observed for these samples, which indicates that the samples had not undergone such changes. Peptide mapping data provided confirmation that the amino acid sequence of each protein sample was the same, as digestion with the same proteolytic enzyme (trypsin) under the same experimental conditions yielded identical fragments (Figure 4).

These data support the assumption that the active ingredient (asparaginase) in both preparations was identical. However, results from circular dichroism analysis and SDS-PAGE suggest that differences exist between the formulations. Very significant differences in CD spectra from 200 to 180 nm were observed (Figures 6 to 8). This could suggest a secondary or tertiary structural change (i.e. how the protein is folded in three-dimensional space), but could also be explained by the presence of far U.V-absorbing excipients in the recombinant formulation, that are not present in the non-recombinant version. This could be further explored by repeating the CD analysis, but this time, removing potential excipients via solid-phase extraction prior to analysis.

Analysis via SDS-PAGE revealed that there were some additional high-molecular mass bands in the non-recombinant asparaginase (with molecular masses ranging from 50 to 100 kDa, by reference to the molecular mass standards) (Figure 1). These may potentially be aggregates of asparaginase (dimers of the asparaginase protein unit). The presence of these aggregates in one formulation but not in the other is potentially due to the differences in formulation that are evident from CD analysis, as some excipients may prevent (or promote) aggregate formation, particularly during storage.

Results from amino acid compositional analysis compared quite well between both asparaginase samples, with 14 out of 17 amino acids having relative standard deviations of less than 15%. However, experimental results did not correlate with the expected composition. This is thought to be due to trace levels of some amino acids, such as glycine, in the reagent-grade chemicals used. Subsequent work on amino acid analysis will attempt to eliminate these sources of contamination. Standard curves for each amino acid have correlation coefficients of > 0.99.

ConclusionsThe impact of formulation excipients on the structural characteristics of biopharmaceuticals should be thoroughly evaluated. A battery orthogonal analytical approaches should be employed, as differences (such as propensity to form aggregates which may impact on product safety or efficacy), may not be readily revealed using techniques such as ‘top-down’ mass spectrometry alone.

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Contact Information

Mary Burke

Managing Director,

Biopharmaceuticals Department,

BioClin Research Laboratories,

IDA Business & Technology Park

Garrycastle, Athlone,

Co. Westmeath

N37 X061

Email: jroche@ait.ie

Derek Bradley (M.Sc.)

Senior Scientist

Biopharmaceuticals Department

BioClin Research Laboratories,

IDA Business & Technology Park

Garrycastle, Athlone,

Co. Westmeath

N37 X061

Email: derek.bradley@bioclinlabs.com

Mary Burke

Managing Director,

Biopharmaceuticals Department,

BioClin Research Laboratories,

IDA Business & Technology Park

Garrycastle, Athlone,

Co. Westmeath

N37 X061

Email: jroche@ait.ie

Derek Bradley (M.Sc.)

Senior Scientist

Biopharmaceuticals Department

BioClin Research Laboratories,

IDA Business & Technology Park

Garrycastle, Athlone,

Co. Westmeath

N37 X061

Email: derek.bradley@bioclinlabs.com