Efficient and Selective Bioconjugation Using Surfactants

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EFFICIENT AND SELECTIVE BIOCONJUGATION USING SURFACTANTS Xi Hu, Thomas Lerch, and April Xu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00594 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Bioconjugate Chemistry

Efficient and Selective Bioconjugation Using Surfactants Xi Hu1*, Thomas F. Lerch2, April Xu1 1Pfizer,

Inc., Biotherapeutics Pharmaceutical Sciences, Worldwide R&D, Pearl River, NY 10965, USA

2Pfizer,

Inc., Biotherapeutics Pharmaceutical Sciences, Worldwide R&D, Chesterfield, MO 63017, USA

ABSTRACT The surfactant sodium decanote is used in the drug substance process of Besponsa, an antibody drug conjugate (ADC), to facilitate bioconjugation between activated calicheamicin derivative (linker payload) and inotuzumab (monoclonal antibody). Under the normal conjugation process conditions, sodium decanoate forms micelles and the micelle formation was shown critical for the efficient conjugation reaction. Further screening studies indicated that sodium dodecyl sulfate, sodium deoxycholate and dodecyltrimethylammonium bromide were also able to facilitate the conjugation reaction. While the choice of surfactant and its concentration in the reaction impact the conjugation efficiency, the charge of surfactant and the choice of linker payload influence the conjugated lysine site selectivity. Eight major conjugated lysine sites are observed in Besponsa, as compared to approximately eighty conjugated lysine sites typically observed in conventional lysine-based ADCs.

INTRODUCTION Surfactants are a general class of organic compounds composed of one polar or ionic hydrophilic end group attached to a non-polar hydrophobic hydrocarbon chain. This amphiphilic property contributes to the unique phase behavior that lowers the surface tension between two phases. Surfactants are widely applied throughout various industries, including detergent, paints, plastics, cosmetics, agriculture and pharmaceuticals.1 In the pharmaceutical industry, surfactants have been mainly used in formulations to improve drug solubility and stability in liquid form, in upstream bioprocessing to enhance protein secretion and in downstream bioprocesses to separate protein from cells and tissues.1-3 This paper describes the use of surfactants in the bioconjugation process of an antibody drug conjugate (ADC). Page 1 ACS Paragon Plus Environment

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Production of antibody drug conjugates involves covalent attachment of a cytotoxic drug (payload) through a reactive linker to an antibody. Compared to small molecule reactions, ADC bioconjugation reactions normally occur at lower concentrations under milder pH and temperature conditions, and that can result in slower reaction kinetics. Often, excess linker payloads are used to drive the reaction to completion and sometimes to compensate for the unstable nature of linker payloads in the reaction media.4-7 When conjugating a hydrophobic linker payload to an antibody, organic solvents such as dimethyl sulfoxide (DMSO), dimethyllformamide (DMF), dimethylacetamide (DMA) and propylene glycol, have been utilized as co-solvents to improve linker payload solubility.5-8 In addition to organic cosolvents and excess linker payload stoichiometry, surfactants were also reported capable of enhancing the bioconjugation efficiency between antibodies and linker payloads, yet the specific role of surfactants in conjugation is not fully elucidated.9-10 Described in this paper is a comprehensive characterization of a surfactant-assisted bioconjugation used for the Besponsa drug substance process, revealing insights into the fundamental mechanism for the efficient and site-selective conjugation observed. Through an improved understanding of the conjugation process, a broad application of this surfactant-assisted conjugation method in bioconjugation area can be expected. Besponsa (inotuzumab ozogamicin) was approved by the U.S Food and Drug Administration (FDA) in 2017 to treat adults with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). It is an antibody-drug conjugate, comprised of a humanized monoclonal antibody (inotuzumab) covalently linked to the antibiotic calicheamicin (ozogamicin).9, 11 Inotuzumab selectively binds to cluster of differentiation 22 (CD22) receptors, which are present on B lymphocytes. Calicheamicin is a potent enediyne cytotoxic agent that causes double-strand breaks in DNA, leading to cell death.12 Besponsa binds to CD22-expressing cells and upon internalization, releases calicheamicin which causes cytotoxicity. Besponsa drug substance, also referred to as the conjugate, is produced from two drug substance intermediates: inotuzumab as the monoclonal antibody and activated calicheamicin derivative (NHS ester of calicheamicin derivative) as the linker payload. The ADC is formed through conventional lysine conjugation chemistry where the succinimidyl-activated calicheamicin derivative reacts with the ε-amino group of lysines on inotuzumab to form a covalent bond. A representation of the drug substance structure is shown in Figure 1.

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Figure 1. Structure of Inotuzumab Ozogamicin Inotuzumab O

Linker

N H

O

N H

CH3 O I O CH3 HO H3CO

CH3 S

OCH3

O

OH

OCH3

O

Calicheamicin

O

N

OH O

O

HO

S

NH

S O CH3 HN HO O N H3CO

O

O

OCH3 O H

O

n~6

The Besponsa conjugation process takes place in basic aqueous media where activated calicheamicin derivative quantitatively reacts with inotuzumab forming the desired ADC within minutes in the presence of sodium decanoate.13 In the absence of sodium decanoate; however, a maximum of 40% inotuzumab is conjugated with activated calicheamicin derivative under the otherwise similar conjugation conditions. Furthermore, unlike conventional lysine-based ADCs that have more than eighty lysine sites modified,14 eight major conjugated lysine sites (four on each half of the antibody) in Besponsa account for more than 90% of the site occupancy. We hypothesized that these unique features of Besponsa could be related to the use of sodium decanoate in the conjugation process. To understand the role of sodium decanoate in the conjugation reaction and the conjugation site selectivity observed in Besponsa, systematic studies were carried out. First, the effect of sodium decanoate on conjugation kinetics and the correlation between sodium decanoate micelle formation and conjugation efficiency were explored. Next, additional surfactants were screened, and their impact on conjugation efficiency was measured at different concentrations. The surfactant-assisted solubilization of activated calicheamicin derivative in conjugation media was assessed. Additionally, the potential for surfactants to perturb inotuzumab structure was tested. Finally, the conjugation site selectivity of Besponsa was compared to the conjugates produced using different surfactants and a linker payload mimic.

RESULTS AND DISCUSSION Surfactant Concentration Impacts Conjugation Efficiency During the development of the drug substance process for Besponsa, multiple process parameters were evaluated in a multi-variant design of experiment (DOE). The results showed that sodium decanoate concentration had a major impact on the conjugation Page 3 ACS Paragon Plus Environment


efficiency, as measured by the amount of unconjugated antibody remaining after conjugation. With increasing sodium decanoate concentration up to 40mM in conjugation reaction, the unconjugated antibody content decreased significantly (Figure 2). Figure 2. Impact of Decanoate Concentration on Conjugation Efficiency

Figure 2-The correlation between the amount of unconjugated antibody at the end of bioconjugation and sodium decanoate concentration in the reaction was pictured in the prediction profiler after DOE analysis using JMP software. The black line represents the predicted unconjugated antibody level at the corresponding decanoate concentration in the reaction, dashed blue lines represent 95% confidence interval for the predicted values. The red dashed straight lines highlight the decanoate concentration used in the normal Besponsa conjugation reaction and corresponding unconjugated antibody level.

To further understand the role of sodium decanoate concentration, conjugations of inotuzumab and activated calicheamicin derivative were conducted in different amounts of sodium decanoate (0mM, 12.5mM, 25mM, 37.5mM and 50mM) and the unconjugated antibody levels were monitored throughout the reactions. The results are summarized in Figure 3, and they show that sodium decanoate concentration impacted both conjugation kinetics and conjugation efficiency. Higher decanoate concentration increased the conjugation kinetics between inotuzumab and activated calicheamicin derivative. The conjugation rate followed 50 mM> 37.5 mM> 25 mM> 12.5 mM> 0 mM. At a 30-minute conjugation time, a larger amount of inotuzumab was conjugated to activated calicheamicin derivative at higher decanoate concentration, consistent with the DOE results. In aqueous solutions, the activated calicheamicin derivative is known to be susceptible to hydrolysis (resulting in inability to conjugate) and only becomes stable when it is covalently attached to inotuzumab.13 Hence, the correlation between conjugation efficiency and sodium decanoate concentration can be mostly attributed to the combined effect of decanoate-enhanced conjugation rate and the limited activated calicheamicin derivative stability. For slower conjugation reactions, the hydrolysis of activated calicheamicin derivative competes with the conjugation reaction, leaving less substrate for conjugation and resulting in a higher amount of unconjugated antibody.


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Figure 3. Sodium Decanoate Affects Conjugation Rate and Efficiency

Figure 3 Five conjugation reactions with various decanoate concentrations under otherwise similar conjugation conditions were monitored for the amount of unconjugated antibody at the designated conjugation time points. Based on the change in unconjugated antibody level over time, the conjugation rate follows 50mM > 37.5 mM > 25 mM > 12.5 mM > 0 mM. Based on the amount of unconjugated antibody at 30 minute reaction time, the conjugation efficiency follows 50mM > 37,5 mM > 25 mM > 12.5 mM > 0 mM.

The results in Figure 3 further demonstrate that the biggest difference in reaction rate and efficiency exists between conjugations with 25mM and 37.5 mM sodium decanoate. Two different behaviors of reaction kinetics were apparent from these data. Under conditions of ≤ 25mM decanoate (0 mM, 12.5 mM and 25 mM), the conjugations between inotuzumab and activated calicheamicin derivative were slow and inefficient. By contrast, fast and quantitative conjugations were achieved in 37.5mM and 50mM decanoate. This distinctive correlation between sodium decanoate concentration and conjugation efficiency is suspected to be related to the sodium decanoate micelle formation. Subsequent studies were carried out to evaluate sodium decanoate aggregate form in the conjugation media. Surfactants form micelles in the conjugation reaction conditions Surfactant molecules are known to self-assemble into micelles when the concentration is above the critical micelle concentration (CMC) and the temperature is above the critical micelle temperature. The literature-reported CMC value for sodium decanoate varies from 40-100 mM 15-16, depending on ionic strength, pH and temperature of the solution. To better understand sodium decanoate micelle formation under the conjugation condition, the CMC of sodium decanoate was experimentally determined. There are multiple methods for determining CMC of a surfactant under a defined condition.15-16 Used here is the dynamic light scattering (DLS) technique. Light scattering intensity is monitored over a range of surfactant concentration and a distinct increase in light scattering intensity corresponds to the formation of micelles starting at the CMC. As shown in Figure 4, the CMC of sodium decanoate under the conjugation condition is around 35 mM, just below the concentration of 37.5 mM described above that afforded an efficient conjugation reaction. Hence it is evident Page 5 ACS Paragon Plus Environment


that sodium decanoate must have formed micelles in both reaction conditions with 37.5 mM and 50 mM decanoate concentration. For the reactions containing 12.5 mM and 25 mM sodium decanoate concentrations, which were below the CMC, the conjugation reactions proceeded slowly and less efficiently in converting inotuzumab to the desired conjugate. Figure 4. Determination of Sodium Decanoate CMC by DLS

Figure 4 – The CMC of sodium decanoate was determined using DLS. Raw light scattering intensity (average derived count rate in counts per second x 1000 (kcps)) was measured as a function of sodium decanoate concentration. The increase in light scattering at ~35 mM sodium decanoate is attributed to micelle formation at this concentration. Triplicate measurements were averaged and plotted with the standard deviations shown as error bars.

To further test the correlation between micelle formation and conjugation efficiency, additional surfactants were screened for their effect on conjugation. Eight surfactants, comprised of anionic (sodium dodecyl sulfate (SDS) and sodium deoxycholate), cationic (dodecyltrimethylammonium bromide (DTAB, or C12TAB) and decyltrimethylammonium bromide (C10TAB)), nonionic (Octyl β-D-glucopyranoside (OGP) and Triton X-100), and zwitterionic (3-(Decyldimethylammonio)propanesulfonate (C10DAPS) and N-Dodecyl-N,Ndimethyl-3-ammonio-1-propanesulfonate (C12DAPS) surfactants, were screened in conjugation reactions between inotuzumab and activated calicheamicin derivative under otherwise similar conjugation conditions. For each surfactant, three different concentrations in the reaction were investigated. Using literature-reported CMC values as guidance, conjugation reactions were carried out to compare surfactants below, at and above their CMCs. At the end of the conjugations, the levels of unconjugated antibody were quantified. The complete list of surfactants, CMC values, the concentrations tested, and the resulting levels of unconjugated antibody are summarized in Table 1. The surfactant was deemed effective in promoting efficient conjugation if less than 10% unconjugated antibody was observed at any one of the investigated concentrations. As shown, SDS at 3 mM, sodium deoxycholate at 6 mM and 11 mM, and DTAB at 25 mM yielded effective conjugation reactions between inotuzumab and activated calicheamicin derivative. The remaining five Page 6 ACS Paragon Plus Environment

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surfactants, at the investigated concentrations, were ineffective in facilitating the conjugation reaction. The lack of effectiveness of these surfactants is speculated to be related to their unfavorable interactions with inotuzumab and/or activated calicheamicin derivative, or the use of sub-optimal concentrations for these conjugation experiments. Table 1. Ionic Type Anionic

Anionic

Cationic

Cationic

Nonionic

Nonionic

Zwitterio nic

Zwitterio

Surfactants Screened In Conjugation Surfactant Name Sodium Dodecyl Sulfate (SDS)

Sodium Deoxycholate

Dodecyltrimethylammo nium bromide (DTAB, C12TAB)

Decyltrimethylammoniu m bromide (C10TAB)

Octyl β-Dglucopyranoside (OGP)

Triton X-100

Literature Reported CMCa 3-16 mM 17-19

Concentration in Conjugation 0.5 mM

Unconjugated Antibodyb 78%

1.5 mM

33%

3 mM

1%

2-8 mM20

1 mM

75%

(5-6 mM)a

6 mM

5%

11 mM

1%

12-20 mM21-23

5 mM

92%

(15-20 mM)a

20 mM

15%

25 mM

6%

10 mM

76%

30 mM

55%

60 mM

51%

10 mM

50%

15 mM

70%

25 mM

63%

0.2 mM

84%

1 mM

90%

2 mM

95%

20 mM

40%

40 mM

61%

60 mM

54%

2 mM

52%

59-68 mM24

1-24 mM25

0.22-0.24 mM26

3(Decyldimethylammonio) propanesulfonate (C10DAPS) N-Dodecyl-N,Ndimethyl-3-ammonio-1-

24-40 mM27

2-4 mM28



nic

propanesulfonate (C12DAPS)

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4 mM

80%

8 mM

85%

The CMC values for sodium deoxycholate (5-6 mM) and DTAB (15-20 mM) were determined experimentally by DLS in the Besponsa conjugation media. a

b

Efficient conjugation conditions (shown in bold text) result in

Efficient and Selective Bioconjugation Using Surfactants

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