Journal of Biotechnology and Bioengineering Volume 1 ... · Reduced Tanon EPS 300 electrophoresis SDS- system Purity Eluted IgG PAGE Tanon 2500 gel Image system Column: TSK G3000SWxl

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Journal of Biotechnology and Bioengineering

Volume 1| Issue 1

Research Article Open Access

Performance of the AbSolute® High Cap resin in the Nimotuzumab capture step

Martinez Rosario 1*, Ruland Yvan 2, Zhao Gang3, and Shen Chunjuan4 1The Center for Molecular Immunology, Cuba. 2, 3, 4, NOVASEP Asia- Biopharma Business Unit, Shanghai, 201203 China. *Corresponding author: Martinez Rosario, The Center for Molecular Immunology, Cuba.

Email: [email protected]

Citation: Martinez Rosario (2017) Performance of the AbSolute® High Cap resin in the Nimotuzumab capture

step: Nessa Journal Biotechnology and Bioengineering.

Received: February 10th 2017, Accepted: March 7th 2017, Published: May 22th 2017

Copyright: © 2017 Martinez Rosario, Yvan Ruland, Zhao Gang and Shen Chunjuan et al. This is an open-

access article distributed under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author and source are

credited.

Abstract

Protein A is the affinity chromatography ligand of choice for first-step capture in the purification of mAbs as its

high selectivity gives excellent purity with high yields. Furthermore,a Protein A capture acts as a key volume

reduction step in the process since it concentrates significantly the product stream. The relatively high cost of

suchaffinity resins leads to consider different operational strategiesin order to well optimize this step. One of

them is to evaluate Protein A matrixes with highperformance in terms of dynamic binding capacity (DBC) at

high flow rates.

Recently a new Protein A ligand, AbSolute® High Cap (AbSolute HC), was introduced by Novasep Company,

which is modified with respect to high binding capacity at higher velocities. This article describes the

performance of AbSolute HC gelfor Nimotuzumab capture, by measuring its Dynamic Binding Capacity (DBC)

at different flow rates and feed concentrations at lab scale. AbSolute HC data are also compared with

MabSelectSuRe, the affinity gel used in the current purification process. The process conditionsare adjusted

including the maximum speed for all steps with an assessment of impact on Nimotuzumab purity using AbSolute

HC. In addition the impact of loading amount on aggregates formation with this media and the resin lifetime are

evaluated. The results show a better DBC at 10% on AbSolute HC than MabSelect Sure, with a higher DBC at

10% on Absolute HC with IgG concentration at 2.3 g/L than 0.13 g/L. No significant loss of purity or yield for

speeds until 1000 cm/h for all steps is observedusing AbSolute HC resin. The lifetime of the media is tested up to

200 cycles with suitable results using NaOH and PAB solutions as CIP buffers.

Key words: Protein A, mAbs, affinity chromatography, AbSolute HC, dynamicbinding capacity (DBC), lineal

flow rate, yield, aggregates.


Journal of Biotechnology and Bioengineering Volume 1| Issue 1

Introduction

The capture step in the downstream processing of monoclonal antibodies (mAbs) is often the bottleneck and the

most expensive step due to the use of Protein A media. Selection of a most suitable affinity resin based on

binding capacity and affinity is typically performed prior to optimization.

In recent years, several new-generation Protein A media have been launched on the market claiming improved

mAb capture, or improved resistance to cleaning agents. AbSolute® High Cap, the new Protein A media by

Novasep, developed for the capture of monoclonal and polyclonal antibodies from large volume fermentation

feedstock, maintains excellent DBCs at high flow rates, therefore providing substantially improved productivity

and strongly reduced costs compared with all existing protein A media.

AbSolute HC is based on a modified silica matrix that exhibits strong mechanical, thermal and chemical

stability. The particles are small (35 μm) with a perfect spherical shape and very narrow size distribution. Pore

size is 100 nm and shows narrow size distribution (± 15 nm). Optimized particles and pores allow high efficiency

and faster mass transfer which increase the Dynamic Binding Capacity (DBC) (1).

Particles Cross Section Surface of Particle

Figure 1: Pictures of AbSolute®High Cap beads

The matrix is rigid and incompressible so a 1L column is packed with 1L of AbSolute HC and the media does

not shrink or swell in different solutions. These exceptional mechanical properties combined with a unique

kinetic performance allow operations at higher velocities (up to 1,500 cm/h, depending on the column size and

equipment).

Its resistance to high pH levels makes it stable through alkaline cleaning and sanitization. Indeed, AbSolute HC

remains stable after 400 cycles of use with alkali washing using 100 mM NaOH + 0.5 M NaCl every 10 cycles(2).

Also Novasep reports that using AbSolute HC gel implies an optimization of the geometry of all columns, thus

minimizing the volume of protein A required to perform the step, in order to maximize productivity. In these

conditions, performing the step at industrial scale using AbSolute® High Cap allows a volume reduction in the

media and an increase in productivity by up to 2.7-fold compared with other industry standards, resulting in a

cost savings of up to $1,000,000 for the first load of media(2,3).



This study outlines the performance of AbSolute HC resin for Nimotuzumab capture by measuring its DBC at

different flow rates and feed concentrations at lab scale. AbSolute HC data are also compared with Mab Select

SuRe, current affinity gel used in the purification process.

The impact of mobile phase velocity and of loading amount on product quality is evaluated as well, with the

product critical quality attributes (CQA’s) being measured by yield, host cell protein (HCP) clearance, and

percent of aggregate.

This report also evaluates the lifetime of the media, tested up to 200 cycles using NaOH and PAB solution

(Phosphoric acid, Acetic acid, Benzyl alcohol mixture). Lifetime is assessed by measuring the variation of DBC

over the time, but product quality in terms of purity is monitored as well over the whole lifetime study.

Materials and Methods

The supernatant is obtained from a perfusion bioreactor of 1000 L of mammalian cell culture. The harvests

filtrated are tested at 0.13 g/L IgG concentration, and at 2.3 g/L IgG concentration after TFF concentration.

Absolute HC and Mab Select Sure resins are packed into columns of 0.5 cm of internal diameter (i.d). Column

height is 10 cm for all experiments except for lifetime study which is carried out with a 5 cm height column.

The equipments used for this study, performed in NovaSep facilities in Shanghai, are listed in the Table 1:

Table 1: List of Equipments used

Equipment Supplier

AKTA Purifier GE Healthcare

Analytical HPLC Waters

Tricorn 5 mm ID columns (5 and 10 cm height) GE Healthcare

Balances Sartorius

SDS Page imaging system Tanon-2005 Tian Neng

Microplate reader Spectramax 190 MD

pH meter/conductimeter Mettler Toledo

Membrane TangenX Novasep

TFF skid Novasep

Buffers used at the different steps are shown in Table 2:



Table 2: Buffers and step in the experimentation

Step Buffer

Loading Nimotuzumab harvests filtrated

Equilibrium 50 mM Tris 150 mM NaCl, pH 7.3-7.5, Cond. 15-20 mS/cm

Washing 1 50 mM Tris 1 M NaCl, EDTA 10mM, pH 7.3-7.5, Cond. 80 - 90

mS/cm

Washing 2 50 mM Tris, 150 mM NaCl, pH 7.3-7.5, Cond 15-20 mS/cm

Elution 0.1 M Citric acid, pH 3.2~3.8, Cond 3-7 mS/cm

Wash Purified water

Cleaning NaOH 100 mM

Storage 20 % ethanol

Experimental Conditions

Analytical Assays:

Table 3 summarizes the analytical assays, samples, methods and equipment used in the experimentation.



Table 3: Analytical assays evaluated

Analytical Sample Method Equipment/column/reagent

items

Feed,

HPLC-RP

Column: Zorbax SB 300 C8,

4.6*250mm,5µm

Flow through/wash

HPLC: waters alliance 2695-996

IgG

for BTC test

Solvents: TFA/ACN; Gradient

concentration

elution

Eluted IgG

UV280

Spectrophotometer

Supplier: APL instrument

Reduced Tanon EPS 300 electrophoresis

SDS- system

Purity Eluted IgG

PAGE Tanon 2500 gel Image system

Column: TSK G3000SWxl

SEC 7.8*300mm

HPLC: Waters Alliance 2695-996

ELISA Kit: Cygnus F220.

HCP Eluted IgG ELISA Equipment: Molecular Devices ,

Spectra Max 190

Protein A ELISA Kit: Cygnus F400

leakage Eluted IgG ELISA Equipment: Molecular Devices ,

Spectra Max 190

All the samples were filtered by 0.2 µm on sterile bottle and stored at 4°C.



Breakthrough curves (BTC’s) at different flow rate – Experimental conditions:

The buffers and steps used for those experiments are listed in table 2 above.

A characterization of the capture step of Nimotuzumab is performed at lab scale from a cell culture supernatant,

by tracing breakthrough curves under different conditions of loading linear velocities.

Feed is loaded continuously onto the column, collections are made at regular interval of time at the column

outlet. The concentration in the breakthrough C is measured by HPLC-RP (see table 3 above) and the dynamic

binding capacities Q (x-axis, mg/mL gel) are calculated using the formula:

Q = C0 * t * F / CV Where:

Q: Dynamic binding capacity (mg/mL resin).

C0: Initial protein concentration (mg/mL).

t: Time (min).

F: Volumetric flow rate (mL/min).

CV: Column volume (mL).

When the concentration C in the breakthrough is approaching the initial concentration C0, the column is fully

saturated. It is washed until the UV baseline is reached and then eluted. A regeneration and equilibration is

performed before the next BTC.

Beside the concentration C in the breakthrough, the concentration is also measured in the Wash stream and

Elution stream.

Impact of speed on purity on AbSolute HC – Experimental conditions:

The buffers and steps used for those experiments are listed in table 2 above. A total of 10 experiments are

performed, by varying the mobile phase velocity between 300 and 1000 cm/h. In some experiments the velocity

is kept the same for all steps (load, wash, elution, CIP), in others the velocity is increased for the wash or elution

step.

The time for loading step is adjusted so that 60 to 80 % of the total capacity previously determined in BTC’s

experiments is reached.

After elution, the product is incubated during 1 hour at low pH condition (elution buffer at pH 3.2-3.8) at room

temperature for viral inactivation. Then a microfiltration using 0.22 µm filter is performed to reduce bioburden

content.



Before SEC, SDS Page, HCP and Protein A analysis (see table 3), the samples collected at elution step are

desalted using following conditions:

Column: Hitrap 5 mL; Resin: Sephadex G 25; Buffer: 20 mM Tris, pH 7.6-8.0, conductivity 0.9-1.2 ms/cm;

Flow rate: 100-150 cm/h; Loading volume: 20-30 % column volume.

Lifetime studies – Experimental conditions:

The lifetime study is carried out on a Tricorncolumn of dimension 5 x 50 mm (GE Healthcare) packed with 1 ml

of AbSolute HC.

200 cycles are performed using the buffer conditions listed in table 2 except the cleaning in place (CIP)

conditions, a mixture of acids and alcohols “PAB” being used instead of NaOH. PAB is a mixture of 120mM

H3PO4, 167 mM acetic acid and 2.2 % benzyl alcohol, applied on the column for cleaning at each cycle with 20

minutes contact time. Every 10 cycles, NaOH CIP is performed as well using a 0.1 mM NaOH solution with 20

minutes contact time as well. When NaOH CIP occurs, the column is washed in between the PAB and the NaOH

cleaning steps. All conditions are summarized in table 4 below.

Table 4: Gel Lifetime conditions used

Step Solution Flow pH Conductivity CV

rate (mS/cm)

PAB PAB: 45 cm/h ~1.5 - 3

Hygienization

[a]

120 mM H3PO4,

167 mM acetic acid,

2.2% benzyl alcohol

NaOH CIP[b] 50 mM Tris-HCl, 150 mM 700 7.3- 15-20 20

/Wash NaCl cm/h 7.5

NaOH CIP[a] 0.1M NaOH, 1M NaCl 60 cm/h 12 - 4

/CIP

[a] PAB hygienization is done for all the 200 cycles. Every 10 cycles, a NaOH CIP is performed as well. Contact

time is 20 minutes in both cases.

[b]Column is washed in between PAB and NaOH CIP.



A series of test is performed to check the column performance over the time, as summarized in table 5 below:

Every 20 cycles starting from cycle 1, a BTC is performed under conditions described previously in this section,

in order to measure the variation of 10 % DBC value along the 200 cycles.

The elution stream is analyzed for Nimotuzumab purity every 20 cycles starting from cycle 20.

The product eluted at cycles 31, 33, 35, 37 and 39 is also analyzed, in order to evaluate the impact on quality of

Nimotuzumab when only PAB solution is used as CIP test, and no NaOH.

Table 5: tests performed during lifetime study with identification of corresponding cycle number

Results and Discussion

BTC test at different flow rate on MabSelect Sure and AbSolute HC.

The quantity of antibodies that can be loaded per mL of gel is determined by the breakthrough curves performed

at the linear velocities that maximize the overall process productivity. The breakthrough curves of the resins

studied are presented in figures 2 and 3 at the linear flow rate defined and feed concentration C0 of 0.13 and 2.3

g/L IgG.



Figure 2: BTC of MabSelect Sure resin at 0.13 and 2.3 g/L IgG in feed.

Figure 3: BTC of AbSolute HC resin at 0.13 and 2.3 g/L IgG in feed.

In all cases, increasing the linear flow rate decreases the dynamic binding capacity. This is mainly due to the fact

that using a faster flow rate increases the resistance to the mass transfer of the protein to the interior adsorption

sites of the matrix beads. Antibodies are very large proteins of low diffusivity in the mobile phase and at higher

speed, a larger number of them will not be able to enter the pores in the matrix beads. As a result of a low

diffusivity, it will also take a longer time for the proteins inside the pores to get out of the bead and undergo

further elution along the column. The higher is the mobile phase speed, the shallower is the adsorption front on

the breakthrough curve and the higher is the concentration of antibody in the breakthrough for a given

loading(4,5).

Numerous chromatographic models have been well described in the literature, to characterize the adsorption

equilibrium and adsorption kinetics (6,7). In the case of AbSolute adsorbent, a modeling approach based on

integration of process modeling and experimentation has been applied and has shown the high capacity

properties of such Protein A adsorbent based on matrix silica (8). As a matter of fact it is remarkable in the figure

3 that the breakthrough curves in AbSolute HC gel are steep whatever the linear velocity, due to a very uniform

particle size distribution and a homogeneous pore size distribution for a better mass transfer.



On the same figure we observe on the BTC’s achieved with MabSelect SuRe resin some difficulties to reach the

full saturation. This has been reported for many other Protein A adsorbents(9) with some suggestions related to

molecular stretching and changes in transport mechanism upon adsorption, broad particle size distribution,

competition between monomers and aggregates. The results as shown on figure 3 could support the explanations

related to particle/pore size distribution and competition between monomers and aggregates. As a matter of fact,

a complete saturation is easier to reach with AbSolute HC material of narrow particles and pores distribution,

and seems to be easier to reach at lower concentration.

Table 6 shows the summary of those results at C/C0 0.1 (Q=10 %).

Table 6: Summary result of dynamic binding capacity (Q) for the resins studied.

MabSelect Sure resin Absolute HC resin

IgG conc. Flow rate Q (g/ L resin) IgG conc. Flow rate Q (g/ L resin)

(g/L) in feed (cm/h) at 10 % C/C0 (g/L) in feed (cm/h) at 10 % C/C0

300 26.8 300 42.2

0.13 600 15.5 0.13 600 36.8

1000 10.7 1000 34.1

300 29.3 300 55.0

2.3 600 14.3 2.3 600 50.3

1000 9.9 1000 42.0

As can be seen better results of dynamic binding capacity are obtained with AbSolute HC gel. The highest values

are observed on Absolute HC with IgG at 300, 600 and 1000 cm/h with a range of 42.2-55.0; 36.8-50.3 and 34.1-

42 g/L depending on IgG concentration in feed at concentration at 0.13 g/L and 2.3 g/L.



Impact of speed on purity on AbSolute HC

Beside the dynamic capacity at high flow rate, the ability of a Protein A affinity media to increase an antibody

purity is obviously of paramount importance in such media performance evaluation.

Table7, figures 4 and 5 show the results achieved in this phase to evaluate the impact of high velocities on

Nimotuzumab purity and yield using different concentrations in the feed. The loading being the longest step in

the process, increasing this step’s speed will have a dramatic impact on the productivity. Three different linear

velocities are tested for loading: 300 cm/h, 600 cm/h and 1000 cm/h.

In some experiments, a higher speed is applied for either the wash step (Exp #4 and #9) or the elution step

(Exp#5 and #10).As a matter of fact, increasing those steps speed will allow to decrease further the cycle time

with some direct impact on production schedules if the method is implemented at manufacturing scale.

Table 7: Summary table of speed impact on purity with Absolute HC

Exp. Flow rate IgG Yield (%) Purity by Purity by

No. (cm/h) conc. HPLC- SDS-

in feed SEC (%) PAGE (%)

(g/L)

1 300 99,23 97,05 94,85

2 600 98,49 97,53 95,1

3 1000 99,06 96,22 95,18

4

300 cm/h: load, elution and CIP;

600 cm/h: wash

0,13

99,08 97,35 96,85

5

300 cm/h: load, wash and CIP; 600

cm/h elution 99,93 97,33 96,28

6 300

2,3

97,91 97,25 96,56

7 600 98,77 97,66 96,54

8 1000 98,93 97,9 96,59

9

300 cm/h: load, elution and CIP;

600 cm/h: wash 96,78 97,63 95,01

10

300 cm/h: load, wash and CIP; 600

cm/h: elution. 98,6 97,7 95,14



Figure 4: Pictures of reduced (right) and non-reduced SDS-PAGE (left) on some experiments.

Figure 5: Chromatogram profile and purity result by HPLC-SEC.

Figure 5: Chromatogram profile and purity result by HPLC-SEC.

As reported in table 5 and figure 4, high purity and recovery values are achieved for each condition evaluated

using Absolute HC resin at both 0.13 and 2.3 g/L IgG concentration on feed (purity being measured by HPLC-

SEC and SDS-Page electrophoresis). No significant loss of purity and yield are observed for speeds until 1000

cm/h for loading, and until 600 cm/h for wash and elution steps (the maximum linear velocity tested for those

steps).

Process-related impurities such as PA leakage and host cell protein (HCP) were also determined in these

experiments. Figures 6 and 7 show the results obtained.

Name Retention

Time

Area %

Area

Height

1 Dimer 6.699 157719 2.14 6963

2 mAb 8.084 7214232 97.70 365039

3 ------ 11.542 12119 0.16 946



Figure 6: PA leakage results at different conditions tested with Absolute HC. For each condition, the left column

shows the result achieved at 0.13 g/L feed concentration, and the right column at 2.3 g/L.

Figure 7: HCP results at different conditions tested with Absolute HC. For each condition, the left column shows

the result achieved at 0.13 g/L feed concentration, and the right column at 2.3 g/L.

According to figures 6 and 7 the Protein A leakage is similar for all the tests performed with different velocities.

There is no significant difference either in HCP values for eluted IgG under various operating conditions tested

with the same concentration of IgG in feed solution.

Some slight difference is observed for HCP content in the tests performed with higher feed concentration (2 g/L)

:HCP is increased when a higher elution speed is used than loading and washing. However such HCP content in

the elution stream is still within specifications typically defined for a Protein A capture step.



Lifetime study

Cleaning, sanitization, and storage procedures that preserve resin function and integrity are essential for

obtaining reasonable resin lifetimes. The behavior of the DBC and purity are evaluated on 200 cycles. Figures 8

shows the results.

Figure 8: DBC on Absolute HC over 200 cycles.

Factors that influence resin lifetime include the nature of the feed stream and raw materials used in its

purification, the resistance of the resin to cleaning and sanitization agents, and proper storage conditions. During

this test, less than 10 % decrease of 10 % BDC is measured after 200 cycles with 0.1M NaOH CIP solution every

10 cycles for 20 minutes contact time per cycle. Beside NaOH CIP every 10 cycles, a PAB hygienization step is

applied as CIP at each cycle, with also 20 minutes as contact time.

Purity and process-related impurities are also analyzed. Figures 9, 10 and 11 show the results.

Figure 9: Purity results by HPLC-RP during 200 Cycles



Figure 10: Protein A leakage results during 200 cycles.

Figure 11: HCP results during 200 cycles.

During the lifetime test, no significant loss of purity is observed over the 200 cycles (figure 9). Protein A

leakage in the elution is stable as well, and even though a slight increase in HCP content is observed, it is

staying within the limits typically defined for this step for such attribute.

Furthermore, as a final observation and as it is mentioned in the experimental section of this article, the eluted

product is also analyzed between two NaOH CIP’s, for cycles 31, 33, 35, 37 and 39 whose CIP step is carried

out with the acidic PAB mixture only. No significant difference is observed regarding purity, HCP and protein

A leakage.



Conclusions

It was shown during the study described in this article that AbSolute HC Protein A media exhibits high

Dynamic Binding Capacities at low and high speed for Nimotuzumab capture. Beside this property of higher

loading, it was verified that product quality stays within specifications of purity, and is not impacted when the

speed of loading step, washing step or elution step is increased.

Both factors capacity and speed will allow to increase the productivity values to be reached with Protein A

media, expressed in terms of quantity of Nimotuzumab produced per Liter of resin per unit of time. Based on

those results, high performances in terms of productivity, purity, yield can be expected from an implementation

at larger scale of this capture step, with some stable results over 200 cycles under the conditions tested in this

study



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https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc28987525_20161015020709.pdf

https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc28987525_20161015020709.pdf

https://www.ncbi.nlm.nih.gov/pubmed/17046339





Documents

Journal of Biotechnology and Bioengineering Volume 1 ... · Reduced Tanon EPS 300 electrophoresis SDS- system Purity Eluted IgG PAGE Tanon 2500 gel Image system Column: TSK G3000SWxl