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ApplicAtion note

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Three’s company: formulation prep and stability screening with Grunt, Lunatic and Uncle

IntroductionThe stability of biologics can be highly dependent on formulation pH. The pH can be screened by buffer exchanging proteins into a variety of buffer salts and running follow-up analytics to determine stability. Current practices for buffer exchange all have limitations. Dialysis requires a large excess of buffer and time. Centrifugal filters and desalting columns are faster, but require more hands-on time. Additionally, all three methods require dilu-tion or concentration steps to reach target protein concentrations. Automating buffer exchange elim-inates these bottlenecks, making it easy to explore a greater experimental space.

Grunt removes the buffer exchange bottleneck (Figure 1) by automating a pressure-based ultra-filtration/diafiltration (UF/DF) method (Figure 2). Buffer exchange is accomplished by repeating cycles of volume measurement, pressurization with gentle orbital mixing and buffer refilling. Grunt, Lunatic and Uncle used in series allows for auto-

mated formulation preparation followed by a quick assessment of protein quality and stability. Lunatic enables concentration, mass recovery and sam-ple quality analysis with only 2 µL of sample in a 16- or 96-well format. Uncle multiplexes stabilitymeasurements including Tm, Tagg, sizing and polydis-

Figure 1: Grunt: an automated formulation preparation and buffer exchange tool.

Figure 2: Grunt uses a pressure based UF/DF method for buffer exchange. Grunt adds protein to each Funl, takes an initial volume measurement and then begins the buffer exchange cycle. After buffer exchange completes an optional concentration step, up to 8-fold, will begin.

STEP 2measureVolume

STEP 3pressure-based

filtration

STEP 4measureVolume

STEP 5Add new

buffer

STEP 1Add protein

STEP 6concentrate

(optional)

protein (mAb, etc) buffer 1 buffer 2

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persity for up to 48 samples at the same time with 9 µL of sample. Scientists can efficiently study a wider range of formulation conditions when auto-mated buffer exchange is combined with small-vol-ume analytical measurements.

This application note demonstrates how Grunt, Lu-natic and Uncle (Figure 3) can be easily combined to create formulations and assess stability. A pH screen from 4.0 to 8.0 was performed using Grunt for buffer exchange and concentrating of a biolog-ic, followed by a formulation quality and stability assessment using Lunatic and Uncle.

Methods A 120 mL stock of 25 mg/mL monoclonal anti-body (mAb1, confidential) in 20 mM sodium suc-cinate, pH 6.3 was prepared for buffer exchange by filtering with a 0.2 µm filter. Twelve buffers were prepared to cover pH 4.0 to pH 8.0 in 0.5 pH unit increments. Four buffers were prepared with 10 mM sodium acetate (Sigma #S1429) and titrated to pH targets 4.0, 4.5, 5.0 and 5.5. 10 mM histidine-HCl (Sigma #H8125) was used to cover pH 5.5, 6.0, 6.5 and 7.0. The final set of four buf-fers was prepared with 10 mM sodium phosphate monobasic (Sigma #795488) at pH 6.5, 7.0, 7.5 and 8.0 (Table 1). Buffer exchange was performed on Grunt at 25 °C with a starting concentration of 25 mg/mL, an initial volume of 8 mL per sample and a final concentration target of 60 mg/mL at

Formulation Prepper

ConcentrationLiberator

StabilityScreener

Figure 3: Grunt, Lunatic and Uncle perform different tasks for biologic analysis. Grunt automates buffer preparation and buffer ex-change. Lunatic performs concentration quantification. Uncle performs numerous screening experiments including thermal melting and aggregation.

3.33 mL per formulation. Grunt was set to target 96% buffer exchange with 50% buffer removal per ultrafiltration cycle and active orbital mixing during the entirety of buffer exchange. The pH was measured with a Mettler Toledo InLab Micro pH electrode. Samples were stored at 4 °C prior to analytical analysis.

Final concentrations and mass recoveries were assessed for each formulation. Concentrations were measured with 2 µL of each sample on a Lunatic with a Lunatic Plate. These concentra-

# Buffer pH

1 10 mM acetate 4.0

2 10 mM acetate 4.5

3 10 mM acetate 5.0

4 10 mM acetate 5.5

5 10 mM histidine 5.5




9 10 mM phosphate 6.5




Table 1: Twelve buffers covering pH 4.0 to 8.0 were screened.

three's compAny: formulAtion prep And stAbility screening with grunt, lunAtic And uncle

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tions were used in gravimetric calculations of mass recovery. All samples were measured in triplicate.

To assess thermal stability and aggregation, the melting temperature (Tm), and aggregation (Tagg) measurements were performed on Uncle. Sam-ples were diluted to 10 mg/mL and 9 µL of each sample was added to a Uni. A Tm/Tagg experi-ment was performed with a temperature ramp of 25 °C to 95 °C with a 0.25 °C/min ramp rate. All samples were measured in triplicate. Data was analyzed with Uncle Analysis Software to rank formulation stability.

Results

Buffer exchangeSetting up buffer exchange on Grunt took under 30 minutes and was guided by a software walk-

through (Figure 4). The software takes users though inputting sample information, adjusting experimental settings and adding reagents to Grunt. Grunt buffer exchanged and concentrated 12 unique mAb1 formulations that covered the pH range of 4.0 to 8.0. The 12 formulations were buffer exchanged and concentrated in 12 hours and 24 minutes, completely unattended.

Concentration and recoveryImmediately following formulation preparation by Grunt, concentrations and mass recoveries were determined by collecting A280 measure-ments on a Lunatic. Sample concentrations were measured without dilution and ranged from 55.3 to 69.9 mg/mL (Figure 5). Mass recoveries were determined using A280 concentration measure-ments and the gravimetrically determined final sample volume. Mass recoveries were above 97% for all formulations (Figure 5).

Figure 4: Grunt runs are set up with a software guided walk through.


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pHThe formulation screen was centered around studying the impact of formulation pH on the stability of mAb1. Prior to assessing stability with Uncle, the pH of each final formulation was mea-sured. All final formulations were measured to have a pH within 0.10 pH units of the target pH (Table 2).

Tm & Tagg

Tm and Tagg measurements were collected in the same experiment over a thermal ramp of 25 °C to 95 °C. The antibody underwent two transitions in each formulation, providing Tm1 and Tm2 values (Figure 6). The pH had a significant impact on the melting temperatures of mAb1. As pH increases,

1 2 3 4 5 6 7 8 9 10 11 12

Formulation1 2 3 4 5 6 7 8 9 10 11 12

Formulation

0

10

20

30

40

50

60

70

80

80

82

84

86

88

90

92

94

96

98

100C

once

ntra

tion

(mg/

mL)

% M

ass

Rec

over

y

Figure 5: A: Final concentrations for each mAb1 formulation. The concentration target was 60 mg/mL. B: Percent mass recovered for each formulation.

Table 2: pH values for each prepared 60 mg/mL mAb 1 sample.

# Buffer pH target Formulation pHΔpH

(formulation – target)

1 10 mM acetate 4.0 4.10 0.10

2 10 mM acetate 4.5 4.57 0.07

3 10 mM acetate 5.0 5.08 0.08

4 10 mM acetate 5.5 5.58 0.08

5 10 mM histidine 5.5 5.57 0.07

6 10 mM histidine 6.0 6.04 0.04

7 10 mM histidine 6.5 6.50 0.00

8 10 mM histidine 7.0 6.98 -0.02

9 10 mM phosphate 6.5 6.54 0.04

10 10 mM phosphate 7.0 6.97 -0.03

11 10 mM phosphate 7.5 7.41 -0.09

12 10 mM phosphate 8.0 7.94 -0.06

A B


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Tm1 trends upward from a low of 58.2 °C at pH 4.0 to 71.4 °C at pH 8.0 (Table 3).

Tagg data was collected simultaneously during the experiment. SLS measurements at 266 nm and 473 nm are sensitive to changes in small parti-cles and larger particles, respectively. Although phosphate formulation Tm values are amongst the

highest, Tagg is several degrees lower compared to histidine and acetate formulations. Aggregation was not observed for several formulations under pH 5.5, but Tm1 values were lower than at higher pH. The intermediate pH range of 5.5 to 7.0 had the highest Tagg (~77–78 °C) and the highest Tm1 (~70 °C). Analyzing Tm and Tagg together suggest-

Figure 6: Melting curves for 12 mAb1 formulations over a 25 °C – 95 °C temperature ramp calculated by the barycentric mean.

Table 3: Tm and Tagg determined by Uncle Analysis software.

# Buffer Tm1 (°C) Tm2 (°C)Tagg (°C)

@ 266 nmTagg (°C)

@ 473 nm

1 10 mM acetate pH 4.0 58.2 74.4 n.d. n.d.

2 10 mM acetate pH 4.5 63.4 77.1 n.d. n.d.

3 10 mM acetate pH 5.0 66.7 78.4 76.0 n.d.

4 10 mM acetate pH 5.5 69.1 79.0 78.0 77.9

5 10 mM histidine pH 5.5 65.7 77.3 n.d. n.d.

6 10 mM histidine pH 6.0 68.4 78.7 76.6 n.d.

7 10 mM histidine pH 6.5 69.9 79.1 78.7 78.8

8 10 mM histidine pH 7.0 70.4 79.2 76.6 77.6

9 10 mM phosphate pH 6.5 70.2 77.8 73.3 75.7

10 10 mM phosphate pH 7.0 70.1 77.6 74.6 74.7

11 10 mM phosphate pH 7.5 70.4 78.4 74.6 72.8

12 10 mM phosphate pH 8.0 71.4 79.4 74.7 75.0


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Unchained Labs 6870 Koll Center Parkway Pleasanton, CA 94566 Phone: 1.925.587.9800 Toll-free: 1.800.815.6384 Email: [email protected]

© 2017 Unchained Labs. All rights reserved. Grunt, Uncle and Lunatic are trademarks and the Unchained Labs logo is a registered trademark of Unchained Labs. All other brands or product names mentioned are trade-marks owned by their respective organizations.

Rev C

ed that mAb1 formulations between pH 5.5 to 7.0 were the most stable.

ConclusionCombining automated buffer exchange with higher throughput analytics makes it possible to rapidly prepare and assess protein formulations. The automated buffer exchange process on Grunt prepared 12 unique pH formulations of mAb1 at 60 mg/mL in an overnight run (<13 hours). To identify the optimal pH range for mAb1, the

protein quality and stability data were quickly acquired with Lunatic and thermal ramp stability measurements (Tm and Tagg) on Uncle. The Grunt/Lunatic/Uncle combination is highly adaptable for work in discovery, analytical and formulation labs, and can be used to increase throughput for construct screens, developability assessment, formulation screens, optimization experiments and other biologic studies.


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