Sreedhar Reddy PhD Thesis soft copy - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8309/9/09_chapter 4.pdf · An aliquot of the cells was seeded back into conical flak with

4 Methodology

General

All the experimental work carried out was in accordance with general cell culture

laboratory procedures (Hartung, 2002 and Davis, 1996).

Biosafety hood was surface sterilized using gauge cloth wetted with IPA (70 %)

and germicidal UV lamp was switched on for 15 minutes along with the 2 L

discard beaker containing 500 mL of 0.1 % sodium hypochlorite (as detailed in

table-77 of appendix) solution for discarding liquid waste (Freshney, 2006).

All solutions / cell culture media to be dispensed were pre-warmed to 37ºC unless

otherwise stated.

Any material taken inside the hood was surface sterilized with 70 % IPA (as

detailed in table-25 of appendix).

Earlier (Sreedhar Reddy, 2008), adherent r-CHO cells (referred as control cells)

were adapted to suspension form (referred as test cells) but the conditions such as

optimal serum percentage in the growth medium and shaker speed (rpm) conditions were

partially optimized. Recombinant EPO produced with suspension adapted cells was

compared with adherent cells

Briefly, control cells were revived propagated and cell banks were prepared

(MCB & WCB). These cells were adapted to suspension form at different serum

concentrations namely, 10, 7.5, 5 & 2.5 % respectively in a conical flasks and incubated

at 37oC in 5 % CO2 atmosphere on an orbital shaker at different speeds namely 150, 300

& 450 rpm respectively.

Finally based on the viability and visual observation, cells maintained with 7.5 %

serum and 300 rpm (referred as test cells) were scaled up and rhEPO was produced.

Parallely, control cells were also treated in a similar manner. A back up bank (3 vials)

was prepared with test cells for further studies.

The rhEPO produced from both adherent (control) and suspension adapted (test)

cells were subjected to SDS-PAGE and RP-HPLC in order to establish the comparison

followed by the assessment of biological activity by in vitro bioassay (Sreedhar Reddy,

2008).

Experimental design for the optimization of serum concentration

With an objective to further narrow down or complete replacement of the serum

concentration in the growth medium, experiments were carried out with reduced serum

conditions and addition of other additional supplements. Figure-1 gives the detailed

experimental design with different medium conditions.

Revival

One of the three vials containing test cells, (Sreedhar Reddy, 2008), that were

cryo-preserved after adaptation to 7.5 % serum concentration at 300 rpm was taken out

form LN2 and slowly thawed for 2 min in the water bath at 37oC. Cells were transferred

aseptically into 15 mL tube with 6 mL growth media containing 7.5 % serum (here after

referred as control growth medium, CGM) (as detailed in table-76 of appendix) and

centrifuged at 1500 rpm for 8 minutes. Pellet was re-suspended in 2 mL of fresh control

growth medium and transferred into T-75 cm2 suspension flask containing 13 mL of

control growth medium. Flask was incubated in an incubator at 37oC in 5 % CO2

atmosphere.

These cells are referred to as control cells and the rhEPO produced from these cells is

referred to as control product.

Cells adapted with the optimized growth medium composition are referred to as test

cells and the rhEPO produced from these cells is referred to as test product

Figure 1: Schematic representation of experimental design for the

optimization of serum concentration & other media components of the growth

medium.

2.5 % Serum Spinner

2 % Serum

Spinner

1.5 % Serum

Spinner

Revival of cells adapted to 7.5% serum (Control cells)

5 % Serum Flask

2.5 % Serum Flask

1 % Serum Flask

5 % Serum

Spinner

2.5 % Serum

Spinner

1 % Serum

Spinner

Discarded on day-6

Discarded on day-3

Discarded on day-6

Discarded on day-20 Discarded

on day-20

Insulin+Pluronic Common Discarded on

day-10 Discarded on

day-6

1 % Serum+ 1.05 mg Insulin+

0.2% F-68

2.5 % Serum+ 525 µg

Insulin+ 0.1% F-68

2.5 % Serum+ no

supplements

2.5 % Serum+ 525 µg Insulin

2.5 % Serum+ 0.1%

F-68

Finalized condition

(Test cells)

Discarded on day-4

Discarded on day-20

Discarded on day-12

Discarded on day-4

An aliquot of the cells was seeded back into conical flak with media containing

7.5 % serum and kept at 300 rpm to serve as back up (control cells).

Medium change was given at every 48 h by pelleting the suspension at 1500 rpm

for 8 minutes and the pellet was gently re-suspended in 75 mL of respective fresh growth

medium. Cells were observed constantly and an aliquot of 1 mL was collected at every

24 h interval until day-4 and at every 48 h interval until day-25 to check the cell viability

and morphology. Cell suspension aliquoted for cell count was replenished with

respective fresh growth medium.

Conical flask with medium composition of 1 % serum + 525 µg Insulin + 0.1 %

Pluronic F-68 was terminated on day-3. Conical flask with 5 % serum + 525 µg Insulin +

0.1 % Pluronic F-68 and spinner with 5 % serum + 525 µg Insulin + 0.1 % Pluronic F 68

were terminated on day-6. Conical flask with 2.5 % serum + 525 µg Insulin + 0.1 %

Pluronic F-68 and spinner with 1 % serum + 525 µg Insulin + 0.1 % Pluronic F-68 was

terminated on day-20. Adaptation was continued with spinner flask containing 2.5%

serum + 525 µg Insulin + 0.1 % Pluronic F-68 until the cell density reached 1 × 106

cells/mL.

On day-25, cells adapted to medium containing 2.5 % serum + 525 µg Insulin +

0.1 % Pluronic F-68 concentration were centrifuged at 1500 rpm for 8 minutes and the

pellet was gently re-suspended in fresh basal medium (DMEM). Cell density was

adjusted to 1 105 cells/ mL and suspended into 250 mL spinner flasks containing

different serum concentrations namely,

2.5 % serum + 525 µg Insulin + 0.1 % Pluronic F-68

2 % serum + 525 µg Insulin + 0.1 % Pluronic F -68 and

1.5 % serum + 525 µg Insulin + 0.1 % Pluronic F-68 (as detailed in table-23 of

appendix), respectively, and incubated at 37oC in 5 % CO2 atmosphere in an orbital

shaker-incubator at 50 rpm

Medium change was given at every 48 h by pelleting the suspension at 1500 rpm

for 8 minutes and re-suspending the pellet in 75 mL of fresh respective growth mediums.

Cells were observed constantly and an aliquot of 1mL was taken at every 24 h interval

until day-4 and at every 48 h interval until day-15 to check the cell viability and

morphology. Cell suspension aliquoted for cell count was replenished with fresh growth

medium.

Spinner flask with 1.5 % serum concentration was terminated on day-6 and

spinner flask with 2 % serum concentration was terminated on day-10. Adaptation was

continued with spinner flask containing 2.5 % serum until the cell density reached 1

106 cells/ mL.

On day-15, cells adapted to 2.5 % serum concentration were pelleted at 1500 rpm

for 8 min and the pellet was gently re-suspended in fresh basal medium (DMEM). Cell

density was adjusted to 1 105 cells/ mL and suspended into 250 mL spinner flasks

containing 75 mL of different combination of mediums namely,

2.5 % serum + 525 µg Insulin + 0.1 % Pluronic F-68

2.5 % serum (without any additional supplements)

2.5 % serum + 525 µg Insulin

2.5 % serum + 0.1 % Pluronic F-68 and

1 % serum + 1.05 mg Insulin + 0.2 % Pluronic F-68 (as detailed in appendix

table-23), respectively and incubated at 37oC in 5 % CO2 atmosphere in an orbital

shaker-incubator at 50 rpm. Medium change was given at every 48 hours by pelleting the

suspension at 1500 rpm for 8 minutes and the pellet was gently re-suspended in 75 mL of

fresh respective growth medium. Cells were observed constantly and an aliquot of 1 mL

was taken at every 24 h interval until day-4 and at every 48 hours interval until day-20 to

check the cell viability and morphology. Cell suspension aliquoted for cell count was

replenished with fresh GM.

Cells with 2.5 % serum concentration (with out supplements) and 2.5 % serum

concentration + 525 µg Insulin were terminated on day-4. Cells with 2.5 % serum + 525

µg Insulin + 0.1 % Pluronic F-68 were terminated on day-12. Adaptation was continued

with spinner flask with 2.5 % serum + 0.1 % Pluronic F-68 until the cell density reached

1 106 cells/mL.

On day-20, cells with 1 % serum + 1.05 mg insulin + 0.2 % Pluronic F-68 were

terminated. Cells adapted to 2.5 % serum + 0.1 % Pluronic F-68 (test cells) were

centrifuged at 1500 rpm for 8 min and the pellet was gently re-suspended in fresh basal

medium (DMEM). Cell density was adjusted to 1 105 cells/mL and re-seeded in the

same medium for scale-up production.

Comparison of productivity and biological activity of control cells (with 7.5 % serum)

and test cells (with 2.5 % serum + 0.1 % Pluronic F-68)

Scale-up

On day-14, cells adapted to suspension form were transferred to one 1L spinner

flask at 1 105 cells/ mL in a total volume of 250 mL of 2.5 % serum + 0.1 % Pluronic

F-68 (optimized growth medium, referred as OGM) at a stirring speed of 50 rpm. After

48 h, 1 mL was aliquoted for cell count and based on the count, cell suspension was

diluted to give 1 105 cell suspension/mL. This was seeded into three, 1L spinner flasks

containing fresh 250 mL OGM. A portion of the cells (test cells) were frozen (as detailed

in table-5 of appendix) in 5 cryovials at a cell density of 1 107 cells/ mL, for later use.

Control (suspension cells adapted to 7.5 % serum) cells were also transferred to

one 1L spinner flasks at 1 105 cells/mL in a total volume of 250 mL of control GM

with a stirring speed of 50 rpm. After 48 h, 1 mL was aliquoted for cell count and based

on the count, cell suspension was diluted (with fresh GM) to give 1 105 cells/mL. This

was seeded into three, 1L spinner flasks containing fresh control growth medium. The

flasks were then incubated in an incubator at 37oC in 5 % CO2 atmosphere.

Production of rhEPO

After 72 h of seeding, suspension adapted (test cells) cells from three spinner

flasks were pelleted in 500 mL centrifuge tubes separately, washed with phosphate

buffer saline (PBS) and pellet from each centrifuge tube was gently re-suspended in 250

mL control production medium (as detailed in table-24 of appendix) and seeded back

into three fresh spinner flasks.

Similarly control cells from three spinner flasks were pelleted, washed with PBS

and the pellet was gently re-suspended in 250 mL control production medium and seeded

back into three fresh spinner flasks.

Production medium was replaced in each spinner flask by centrifuging and

suspending the pellet gently in to fresh medium for every 24 h until day-6. Both the cells

were aliquoted for cell counts and viability.

Collection of harvest

After 48 h of incubation with production medium and up to day-6, supernatant

form six spinner flasks were collected into the centrifuge tubes and centrifuged to

remove the cell debris. The supernatant was subjected for pH check, membrane filtered

and stored at -20oC.

Protein Analysis

To check the identity, purity and quantity of rhEPO elaborated by the control

(suspension adapted with 7.5 % serum concentration) and test cells, samples (suspension

cells with 2.5 % serum + 0.1 % Pluronic F 68) were subjected to SDS-PAGE, RP-HPLC.

To check the biological activity (dose - response), in vitro assay (bioassay) using TF-1

(Borellini and Glazer 1993) cells was performed. (Methodology for protein analysis was

mentioned under protein characterization of methodology section).

Process Optimization for the Production of rhEPO

With an objective to attain the maximum yield (rhEPO), experiments were

performed with the test cells under two different phases.

Phase-I: Optimization of production medium composition.

Phase-II: Optimization of production parameters (pH, temperature and percent

CO2).

Two level full factorial design was used at both the phases using Design expert

software (DOE) (Stat-ease Inc, Minneapolis, MA, USA). The design suggests the

interaction of different parameters in the same experiment, minimizes the number of

experiments to be performed and also suggests the run order to be followed while

performing the experiments. In both the phases, optimum parameters or growth

conditions were concluded with the rhEPO yield obtained, which was quantified by

using RP-HPLC.

Revival

One of the vials from the cell bank (test cells) which was frozen after complete

adaptation of suspension cells in OGM was removed from LN2, and slowly thawed for 2

minutes in the water bath at 37oC. Cells were transferred aseptically into 15 mL tube

containing 6 mL OGM and centrifuged at 1500 rpm for 8 minutes. Pellet was gently re-

suspended in 10 mL of fresh OGM and transferred into 250 mL spinner flask containing

75 mL fresh OGM. For cell count, 500 µL media containing the suspended cells were

aliquoted. Flask was incubated in an incubator at 37oC in 5 % CO2 atmosphere with

impeller speed set at 50 rpm.

Propagation

After three-days of incubation, cells were centrifuged at 1500 rpm for 8 min.

Pellet was gently re-suspended in 10 mL of fresh OGM and the suspension was seeded

uniformly into four spinner flasks containing 75 mL of OGM and incubated in an

incubator at 37oC in 5 % CO2 atmosphere.

Phase-I: Optimization of production medium composition

All the four critical components were considered as the optimization parameters

(Figure 2) and an experiment was designed using 2 level full factorial design (Figure 3)

with present component compositions as medium levels and a level below and above the

centre points. Both lower and upper levels were assigned based on the information from

the reference articles or equal ratio (Table 1). Experimental run order and the

combination of the parameters were based on the software prediction (Figure 4).

Figure 2: Computer screen display of design expert software highlighting the selected

factorial method (2-level factorial) and the number of factors (24)

Figure 3: Design of experiments (DOE) computer screen display of 2-Level Factorial

design with details of four different factors, units along with low and high level values.

Table 1: Low level, medium level and high levels of four different parameters that were

selected as a part of optimization of production medium composition.

Parameter Low Level Medium Level High Level

Non Essential Amino acids (NEAA), 100X

(mL) 1.0 3.0 5.0

Insulin, 35mg/10mL (µL)

25.0 150 275

D-Glucose (g) 2.25 4.5 6.75

L-Glutamine, 100 mM (mL)

4.0 6.0 8.0

Figure 4: Screen shot of the run order along with the details of combinations of four

different factors in the production medium suggested by the design of experiments

(DOE) software.

For the 4 factors considered, software suggested 38 experiments, which are to be

performed on two different days. Thus, for convenience, experiments were divided into 2

blocks and were performed in suspension specific 6 well plates. Model suggested total

38 experiments with 3 centre points with 2 replicates. Recombinant EPO yield for each

experiment was quantified using RP-HPLC as a response evaluating parameter to

conclude the optimized condition.

Cell Seeding

After four days of incubation, cell suspension from the entire spinner flasks were

pooled and centrifuged at 1500 rpm for 8 min. Pellet was gently re-suspended in 50 mL

of fresh OGM, from which 1 mL suspension was aliquoted for cell count.

Cell density was adjusted to 1 105 cells/ mL and dispensed 4 mL into seven

suspension specific 6 well plates. To serve as back up 15 mL was seeded back into fresh

spinner flask and maintained at low density. Back up flask and the experimental plates

were placed on an orbital shaker at 50 rpm and incubated in an incubator at 37oC in 5 %

CO2 atmosphere.

After 24 h of seeding in the 6 well plates, all plates were centrifuged at 2000 rpm

for 10 minutes, aspirated the supernatant medium gently and 4 mL of production

medium supplemented with different concentrations of NEAA, Insulin, D-Glucose and

L-Glutamine (as detailed in table-24 of appendix).

Collection of Harvest

After 48 h of incubation with production medium to day-6, supernatant from all 6

well plates were collected daily into 15 mL centrifuge tubes and replenished with fresh

production medium. The collected supernatant was centrifuged to remove the cell debris

and pH was recorded. After membrane filtration, supernatant was stored at -20oC.

Morphology, appearance of cells, condition were monitored though out the collection

process. Cell count and viability check were not performed due to limitation cell volume.

Protein (rhEPO) Analysis

The amount of rhEPO was quantified by RP-HPLC from the supernatant

collected from the 38 different experiments.

Phase II: Optimization of production parameters

Using the medium composition that was optimized during phase-I, different

growth conditions like temperature, pH and percent CO2 at three different levels were

optimized in phase-II. Temperature and percent CO2 levels were maintained with the

help of incubator and the pH levels were maintained in the medium. Similar to phase-I,

rhEPO yield was considered as response criteria for evaluating the optimized parameter.

For the three factors considered (Table 2), along with the centre, medium and high levels

the soft ware suggested a total of 29 experiments (Table 3).

Table 2: Low, Medium and high levels of three different parameters that were selected

as a part of optimization of production parameters.

Parameter Low Level Medium Level High Level

pH 7.0 7.2 7.4

CO2 (%) 4.0 5.0 6.25

Temperature (oC) 35.5 37 38.5

Table 3: Representation of run order along with the details of combination of three

different factors.

Experiment Number pH CO2 (%) Temperature

(oC) 1 7.0 4.0 37.0

2 7.0 5.0 37.0

3 7.0 6.25 37.0

4 7.2 4.0 37.0

5 7.2 5.0 37.0

6 7.2 6.25 37.0

7 7.4 4.0 37.0

8 7.4 5.0 37.0

9 7.4 6.25 37.0

10 7.0 5.0 37.0

11 7.0 4.0 35.5

12 7.0 5.0 35.5

13 7.0 6.25 35.5

14 7.2 4.0 35.5

15 7.2 5.0 35.5

16 7.2 6.25 35.5

17 7.4 4.0 35.5

18 7.4 5.0 35.5

19 7.4 6.25 35.5

20 7.0 5.0 37.0

21 7.0 4.0 38.5

22 7.0 5.0 38.5

23 7.0 6.25 38.5

24 7.2 4.0 38.5

25 7.2 5.0 38.5

26 7.2 6.25 38.5

27 7.4 4.0 38.5

28 7.4 5.0 38.5

29 7.4 6.25 38.5

Due to the limitation of availability of the incubator, followed one factor at a time

(OFAT) instead of two factorial method in Phase-II was adopted. A total of 29

experiments were executed in three steps

Step-1: Plate A (Experiment 1, 4 & 7)

Plate B (Experiment 2, 5, 8, 10 & 20)

Plate C (Experiment 3, 6 & 9)


Plate B (Experiment 12, 15 & 18)



Plate B (Experiment 22, 25 & 28)


Cell Seeding for step-1 experiments

The cell density of the cell suspension that was maintained as back up in a

spinner flask was centrifuged at 1500 rpm for 8 min. pellet was gently re-suspended in

50 mL of fresh OGM, from which 1 mL suspension was aliquoted for cell count. Cell

density was adjusted to 1 105 cells/ mL in the respective pH mediums and dispensed 4

mL into three (Plate A to C) suspension specific 6 well plates. Plates were placed in

different incubators with different (Plate A at 4 %, Plate B at 5 % and Plate C at 6.25 %)

CO2 levels in an orbital shaker incubator at 50 rpm at 37oC.


Pellet that was re-suspended for step-1 experiments was used here. Cell density

was adjusted to 1 105 cells/ mL in media with different pH and later 4 mL was

dispensed into three (Plate A to C) suspension specific 6 well plates. Plates were placed

in different incubators with different (Plate A at 4 %, Plate B at 5 % and Plate C at 6.25

%) CO2 levels on an orbital shaker incubator at 50 rpm at 35.5oC.


Pellet that was re-suspended for step-1 experiments was used here. Cell density

was adjusted to 1 105 cells/ mL in the media with different pH and later 4 mL was

dispensed into three (Plate A to C) suspension specific 6 well plates. Plates were placed

in different incubators with different (Plate A at 4 %, Plate B at 5 % and Plate C at 6.25

%) CO2 levels on an orbital shaker at 50 rpm at 38.5oC.

To maintain as back up, 25 mL of the suspension was seeded back into fresh

spinner flask and maintained at low density and placed in an orbital shaker-incubator and

incubated at 37oC in 5 % CO2 atmosphere.

Optimization

After 24 hours of seeding in the multi-well plates, all the plates were centrifuged

at 2000 rpm for 10 minutes and the spent medium was aspirated, which was

supplemented with 4 mL of production medium with respective pH (media composition

remains same as optimized at phase-1) and placed on an orbital shaker. Plates are

incubated at respective incubators.


After 48 h of incubation with production medium to day-6, supernatant was

collected daily into 15 mL centrifuge tubes and centrifuged to remove the cell debris.

The pH of the supernatant was recorded and later membrane filtered and stored at -20oC.

Morphology and appearance of cells were monitored though out the collection process.

Cell count and viability check were not performed due to limitation of the total cell

volume.


The amount of rhEPO from all the experiments with different parameters was

quantified by RP-HPLC.

Scale -up with the Optimized Production Medium and Parameters

Propagation of Cells

Cells that were maintained as back up were propagated further in OGM and

seeded in to three 1 liter spinner flasks.

When the cell density reached 1 106 cells/mL, cells were centrifuged at 1500

rpm for 8 min. Pellet was re-suspended in fresh optimized production medium, OPM (As

detailed in table-9 of appendix) and incubated at optimized conditions (pH, 7.2; CO2, 5

% and Temperature, 35.5oC).

After 24 h of incubation at the optimized condition, media change was given with

fresh optimized production medium.


After 48 h of incubation with optimized production medium to day-6, supernatant

from all three spinner flasks were collected by pelleting the cells. Fresh optimized

production media was replenished. The pH of the supernatant was recorded and later

membrane filtered and stored at -20oC. Cell count and viability check of cells were

performed though out the collection process.


All the harvests were pooled and the amount of rhEPO in the harvest was

quantified by RP-HPLC in triplicates.

Protein Purification (Down Stream Processing)

Recombinant Human Erythropoietin (rhEPO) secreted by recombinant Chinese

Hamster Ovary (r-CHO) cells into the medium referred to as harvest. This harvest of

rhEPO contains impurities (product related or non-related) and cell culture media

components. The rhEPO produced by these cells posses varying degree of glycosylation

that determines the biological activity of rhEPO. Through downstream process (DSP)

using different chromatography techniques, the rhEPO in the harvest was purified to

remove impurities and to obtain rhEPO with desired isoforms (as a function of

glycosylation) meeting final product specifications.

The harvest collected from all the three spinner flasks after scale up using

optimized parameters was pooled (total ~3600 mL) and subjected to purification process

as outlined in Figure 5. The list of buffers used for storage, sanitization, regeneration,

equilibration, washing and elution at different purification steps are outlined in Table 4.

Basically any cell culture harvest containing extra cellular protein are subjected

to purification in 3 steps (www.biotecharticles.com/Others-Article/Three-Phase-Protein-

Purification-Strategies-386.html) namely,

1. Capturing

2. Intermediate purification and

3. Polishing.

Harvest

Affinity Chromatography

Dia filtration

Cation Exchange Chromatography

Anion Exchange Chromatography-I

Anion Exchange Chromatography-II

Gel Filtration Chromatography (GFC)

Membrane filtration (0.2 µm)

Figure 5: Flow Chart of Purification steps

Table 4: Application and composition of different buffers used in rhEPO purification

In the present study, the same principle was applied. Affinity chromatography

served as capture step wherein all the unwanted proteins present in the harvest were

separated from target protein, which is captured by the affinity matrix. Cation exchange

and Anion exchange chromatography-I served as intermediate purification steps, where

protein related and differently charged impurities were removed. Anion exchange

chromatography-II served as polishing step by concentrating the volume as well as

removing the aggregates generated during intermediate purification. Dia-filtration and

Process

step

Application

Storage Sanitization Regeneration Equilibration Washing Elution


20 % Ethanol + 0.1M

KH2PO4, pH 8.0

0.1 M NaOH Nil PBS, pH 7.2 Nil PBS + 0.9 M NaCl, pH 7.2

Dia-filtration

0.1 M NaOH 1 M NaOH Nil 20 mM Tris, pH

7.0 Nil Nil


0.01 M NaOH 1 M NaOH

20 mM Tris+ 1.4 M NaCl, pH

8.0

20 mM Tris, pH 7.0 Nil Nil

Anion Exchange Chromatography-I

0.01 M NaOH

1 M NaOH + 1 M NaCl

20 mM Tris+ 1.4 M NaCl, pH

8.0

20 mM Tris, pH 8.0

20 mM Tris+ 70

mM NaCl, pH 8.0

20 mM Tris+ 140 mM NaCl,

pH 8.0

Anion Exchange Chromatography- II

0.01 M NaOH

1 M NaOH + 1 M NaCl Nil 50 mM Tris, pH

8.0

20 mM Sodium Acetate,

pH 6.0 and 3.7

20 mM Tris+ 1 M NaCl, pH

8.0

Gel Filtration Chromatography

0.01 M NaOH 0.5 M NaOH Nil 20 mM Sodium

acetate, pH 6.00 Nil Nil

Membrane Filtration (0.2µm)

Nil Nil Nil Nil Nil Nil

gel filtration played major role in exchanging the desired product to the desired buffer by

removing salts and chromatophores. Finally, membrane filtration ensured the absence of

foreign contaminants and enabled the storage of purified protein in aseptic conditions.

Chromatography system

Automated chromatography system (AKTA Basic & AKTA Explorer from GE

Health Care, Chennai, India) integrated with UV, pH and conductivity monitors was

used for control and monitoring of rhEPO purification process. With its integrated

Unicorn software (GE Health care, Chennai, India), automated chromatography system

has advantages such as real time control and evaluation of process, automation of run

using programming, archiving and storing of run data. It also enables off-line analysis

and evaluation of data and report preparation. Due to high volumes, affinity

chromatography, cation exchange chromatography and anion exchange chromatography-

I were processed on AKTA purifier as it enables loading of input at high flow rate (> 100

mL/minute). With an objective to reduce the holdup volume, AKTA Basic was used for

anion exchange chromatography-II and gel filtration chromatography.

Note: - Fraction collection in all the purification steps is based on the UV. All the

fractions were collected between the desired UV range i,e., from the time peak started

and till it reached back to the desired UV. This is applicable to all the further sections.

Note: - Fractions that were collected during all purification steps was based on UV

absorption at 214 nm.


Principle

Affinity (pseudo) Chromatography separates proteins on the basis of a reversible

interaction between a protein (or group of proteins) and a specific ligand attached to a

chromatographic matrix. The technique is ideal for a capture or intermediate step and can

be used whenever a suitable ligand is available for the protein(s) of interest. Here most of

the unwanted proteins will be removed in flow through retaining the desired protein.

Role of the step

This step involves the removal of colored impurities, unwanted proteins and to

reduce the volume by concentrating the harvest for subsequent steps. Figure 6 shows the

key stages of affinity chromatography and Figure 7 shows a typical chromatogram of

affinity chromatography.

Resin used

Blue Sepharose FF (fast flow) (GE Health care, Chennai, India)

Protocol conditions

Load pH: 7.2 ± 0.2

Conductivity: 16.5 ± 1 mS/cm.

Protocol parameters

The pH of the harvest was adjusted to 7.2 with HCl/ NaOH.

The storage solution was removed from the column, and pre equilibrated with PBS,

pH 7.2 (as detailed in table-1 of appendix).

Harvest was loaded with a flow rate of 225 mL/minute

The bound protein was eluted using 40 % gradient with the elution buffer, PBS + 0.9

M NaCl, pH 7.2 (as detailed in table-56 of appendix)

Output fraction containing the bound protein was collected when the absorbance

reached 5 mAu and dropped down to 20 mAu.

The column was cleaned with 1 M NaOH (as detailed in table-54 of appendix),

sanitized the column with 70 % IPA (as detailed in table-57 of appendix) and stored

with 0.1 M KH2PO4 + 20 % IPA (as detailed in table-58 of appendix)

An aliquot of 1 mL of the output was restored for analysis by RP-HPLC, SDS-PAGE

and IEF.

Figure 6: Key stages in Affinity Purification (http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/0E9DAB3298C9C74AC125762800

1CCDD6/$file/18102229AE.pdf)

Figure 7: A typical chromatogram of Affinity purification (http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/0E9DAB3298C9C74AC125762800

1CCDD6/$file/18102229AE.pdf)

Dia-filtration

Principle

Dia-filtration or Cross flow filtration or Tangential flow filtration (Figure 8) is

different from dead-end filtration (Figure 9) in which the feed is passed through a

membrane or bed, the solids being trapped in the filter and the filtrate being released at

the other end. In, cross-flow filtration majority of the feed flow travels tangentially

across the surface of the filter, rather than into the filter, hence the name cross-flow

filtration. The main advantage of cross-flow filtration is that the filter cake (which can

blind the filter) is substantially washed away during the filtration process, increasing the

length of time that a filter unit can be operational. Moreover, it can be a continuous

process, unlike batch-wise dead-end filtration.

Role of the step

This step is used for buffer exchange and removal of chromatophores present in

the production medium. This also removes low molecular weight impurities (< 10 kDa).

Membrane used

Sortocon, 0.7 m2, 10 kDa cut off (Sartorius AG, Goettingen, Germany).

Protocol parameters

The cassette was removed from the storage solution and assembled with the

membrane in between the gaskets. Tubing was arranged (one tube for loading, one

tube for permeate and one for retentate). Both loading and retentate tubing’s were

placed in the container holding the harvest where as the permeate tube was placed in

the waste collecting container. Pressure knob was adjusted to achieve a flow rate of

800-900 mL/minute.

Cassette was equilibrated using 20 mM Tris, pH 7.0 (as detailed in table-59 of

appendix) and the output obtained after affinity chromatography was concentrated 4

fold by passing the output through membrane and collecting the retentate. This

Figure 8: Diagrammatic representation of Tangential flow filtration

(http://www.pall.com/laboratory_34212.asp)

Figure 9: Diagrammatic representation of Dead End Filtration (http://www.pall.com/laboratory_34212.asp)

process was repeated twice and retentate was collected by flushing air through feed

(tube for loading).

Then the membrane was sanitized with 1 M NaOH (as detailed in table-54 appendix)

and incubated for one hour.

After one hour, 1 M NaOH was removed and the membrane was stored with 0.1 M

NaOH till further use.


and IEF.

Ion Exchange Chromatography

Principle

In ion exchange chromatography retains analyte molecules based on columbic

(ionic) interactions on the column. Ionic functional groups are displayed on the

stationary phase surface that interacts with analyte ions of opposite charge. This type of

ion exchange chromatography is further subdivided into cation exchange

chromatography and anion exchange chromatography. The ionic compound consisting of

the cationic species (positively charged) and the anionic species (negatively charged) can

be retained by the stationary phase. Figure 10 shows the mode of binding of differently

charged particles on a resin.

Cation exchange chromatography retains positively charged ions because the

stationary phase displays a negatively charged functional group where as anion exchange

chromatography retains anions using positively charged functional group


Role of the step

This step is primarily useful for the removal of low glycosylated and some non-

glycosylated EPO along with host cell proteins. EPO with desired glycosylation comes

in flow through. Figure 11 shows a typical chromatogram of ion exchange

chromatography.

Figure 10: Principle of ion exchange chromatography: Mode of binding of differently

charged particles on a resin (http://www.ucl.ac.uk/~ucbcdab/enzpur/ionX.htm)

Figure 11: Typical chromatogram of Ion Exchange Chromatography (http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/3E56FFCAFE43BCADC12576280

01D0EC6/$file/11000421AA.pdf)

Resin used

SP (Sulfo Propyl) Sepharose FF (fast flow) (GE Health care, Chennai, India)

Protocol conditions

Load pH: 7.0 ± 0.2

Conductivity: 3.0 ± 0.5 mS/cm

Protocol parameters

Storage solution was removed from the column and pre-equilibrated the column

using equilibration buffer, 20 mM Tris pH 7.0 (as detailed in table-59 of appendix)

Dia-filtration output was loaded at 225 mL/minute and the flow through was

collected between the absorbance, 5 mAu and 10 mAu.

After flow through collection, column was regenerated with 20 mM Tris + 1.4 M

NaCl, pH 8.0 (as detailed in table-60 of appendix).

Column was sanitized with 1 M NaOH (as detailed in table-54 of appendix) and

stored with 0.01 M NaOH


and IEF.

Anion Exchange Chromatography-1

Role of the step

This step mainly removes the impurities and aid in intermediate purification.

Resin used

Q-Sepharose FF (GE Health care, Chennai, India)

Protocol conditions

Load pH: 8.0 ± 0.2

Conductivity: 1.7 ± 0.2 mS/cm.

Protocol parameters

Storage solution of the column was removed and equilibrated with 1 M Tris (as

detailed in table-61 of appendix)

The pH of Cation exchange chromatography flow through was adjusted to 8.0 using

equilibration buffer, 1 M Tris followed by loading it to column.

Column was washed with 70 mM salt and the protein was eluted using 140 mM salt.

Peak was collected between the when the absorbance was between 5 mAu to 3 mAu.

Column was regenerated with 20 mM Tris+1.4 M NaCl, pH 8.0 (as detailed in table-

60 of appendix).

Column was sanitized with 1 M NaOH + 1 M NaCl and then stored with 0.01 M

NaOH.

1 mL of the output was restored for analysis by RP-HPLC, SDS-PAGE and IEF.

Anion Exchange Chromatography-II

Role of the step

This step is primarily used as a polishing step to remove low glycosylated

isoforms and to concentrate the product. Figure 12 shows a typical chromatogram of a

polishing step.

Resin used

Q-Sepharose FF (GE Health care, Chennai, India)

Protocol conditions

Load pH: 8.0 ± 0.2

Conductivity: 3.0 ± 0.7 mS/cm

Figure 12: A typical chromatogram of polishing step (http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/3E56FFCAFE43BCADC12576280

01D0EC6/$file/11000421AA.pdf)

Protocol parameters

Load (anion exchange chromatography-I output) was diluted 5 times with WFI to

bring down the conductivity to 3.0 ± 0.7 mS/cm

Storage buffer was removed and equilibrated the column with pre-equilibration

buffer, 50 mM Tris, with pH 8.0.

Anion exchange chromatography-I output was loaded at a flow rate of 25 mL/minute

and the column was washed with Tris buffer (as detailed in table 61 of appendix).

Then the column was washed with Tris buffer to increase the pH

Elution buffer (1 M NaCl) was used and 10 mL/min fractions were collected.

Fraction collection is between 5 mAu and 4 mAu.

Column was sanitized with 1 M NaOH + 1 M NaCl and stored at 0.01 M NaOH


and IEF.

Gel Filtration

Principle

Size exclusion chromatography (SEC) is a chromatographic method in which

molecules in solution are separated based on their size (more correctly, their

hydrodynamic volume).

Role of the step

This step is mainly used for desalting and exchange of the desired protein into a

new buffer (formulation buffer) based on the difference in the molecular weight of

protein and salt. Figure 13 shows a typical chromatogram of desalt/buffer exchange.

Figure 13: A typical chromatogram of Buffer exchange or Desalting of protein

(http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/226C564DBB7D5229C125762800

1CD36F/$file/18102218AK.pdf)

Resin used

Sephacryl S-200 HR

Protocol conditions

Load pH: 6.9 ± 0.3

Conductivity: 14.5 ± 0.5 mS/cm.

Protocol parameters

Storage solution was removed and equilibrated the column with formulation buffer

(as detailed in table-72 of appendix).

Output obtained from the anion exchange chromatography-II was loaded at 25

mL/minute flow rate and the fractions were collected between 5 mAu and 4 mAu.


and IEF.

Membrane Filtration (0.22 m)

Role of the step

This step is mainly used to filter the sample and to ensure that protein is devoid

of any foreign contaminant.

Filter used

Polyether sulfone low protein binding (Pall Acro disc, PALL India Pvt Ltd,

Mumbai, India)

Protocol parameters

Biosafety hood was switched on and surface sterilized with 70 % IPA (as detailed in

table-25 of appendix).

Sterile 50 mL centrifuge tube, 50 mL syringe, 0.22 µm filter and the tube holding the

gel filtration output were surface sterilized with 70 % IPA and placed inside the

laminar air flow. Filter was fitted to the syringe and was rinsed with formulation

buffer to perform pre-integrity test.

Then the anion exchange chromatography-II output was filtered and the output was

collected into a sterile 50 mL tube.

An aliquot of 1 mL was collected for further analysis and rest of the protein was

stored at -20oC.

Protein Characterization

All rhEPO samples that were generated at the time of comparison of control cells

(suspension adapted cells with 7.5 % serum) and test cells (suspension adapted cells in

2.5 % serum + 0.1 % pluronic F 68) were initially subjected to RP-HPLC. Fractions were

collected into 0.6 mL micro-centrifuge tubes for analysis by SDS-PAGE and in vitro

bioassay.

The amount of rhEPO present in the samples that were generated while scale up

and process optimization were determined by RP-HPLC analysis with an objective of

selecting high protein yielding production medium composition along with production

parameters namely, temperature and pH.

The fractions collected (output or flow through) collected during different steps

of the purification process were subjected to rhEPO quantification by RP-HPLC, SDS-

PAGE and IEF.

Final purified protein was extensively characterized by using different analytical

tools viz,

(i) Iso electric focusing (IEF) for isoform distribution,

(ii) Western blotting, Peptide mapping and N-terminal sequencing for

identity,

(iii) Estimation of Sialic acid to check the extent of glycosylation,

(iv) SDS-PAGE for molecular weight determination

(v) RP-HPLC for quantification and to check the purity of the protein.

(vi) Finally the biological activity of the purified product was determined by

the in vitro bioassay using EPO responsive cells TF-1 and compared

against EPO WHO International standards (NIBSC) and Commercial

EPO Standard (EPREX).

Note: At the time of protein characterization of the final purified rhEPO produced by test

cells (suspension adapted with 2.5 % serum+0.1 % pluronic F-68 followed by process

optimization), pooled harvest of the control cells (adherent cells) was also analyzed by

RP-HPLC for establishing the comparison.

RP-HPLC

Reversed phase HPLC (RP-HPLC) consists of moderately polar mobile phase

and a non-polar stationary phase. Hence, the retention time is longer for molecules which

are more non-polar in nature, allowing polar molecules to elute more readily. Addition of

polar solvent to the mobile phase increases the retention time and decreases by the

addition of more hydrophobic solvent.

The characteristics of the analyte molecule play an important role in its retention

characteristics. In general, an analyte with a longer alkyl chain length results in a longer

retention time because it increases the molecule's hydrophobicity. However, very large

molecules can result in incomplete interaction between the large analyte surface and the

alkyl chain. With increase in hydrophobic surface area retention time also increases

which is roughly inversely proportional to solute size. As the overall surface area

decreases Branched chain compounds elute more rapidly than their corresponding

isomers.

Principle

Reverse Phase-HPLC operates on the principle of hydrophobic interactions which

result from repulsive forces between a relatively polar solvent, the relatively non-polar

analyte, and the non-polar stationary phase and the driving force in the binding of the

analyte to the stationary phase is the decrease in the area of the non-polar segment of the

analyte molecule exposed to the solvent. The decrease in free energy from entropy

dominates hydrophobic effect which is associated with the minimization of the ordered

molecule-polar solvent interface and the hydrophobic effect is decreased by adding more

non-polar solvent into the mobile phase. This mechanism shifts the molecules partition

coefficient such that the analyte spends some portion of time moving down the column

in the mobile phase, eventually eluting from the column.

Chromatographic conditions

Flow rate 1 mL/ min Temperature 250C Detector (UV) 215 nm Pressure 250 bar Sample volume 100 µL

Equilibration of column

Time (min)

Mobile phase A, % (100 % water for injection and 0.1% Tri

fluoro acetic acid (TFA)

Mobile phase B, % (100 % Acetonitrile

and 0.1% TFA) 0 min 60 40 2 min 60 40 20 min 0 100 22 min 0 100

Gradient details

Time (min)

Mobile phase A, % (100 % WFI and 0.1 % TFA)

Mobile phase B, % (100 % Acetonitrile + 0.1 % TFA)

0 min 60 40 2 min 60 40 5 min 0 100 8 min 0 100 11 min 60 40 15 min 60 40

SDS-PAGE

The electrophoretic method reported specifically for EPO analysis was used in

the present investigation (European Pharmacopoeia).

Glass plates (160 x 160 mm), spacer (1-mm thick with 8-well comb), were used

for electrophoresis. For protein separation, 12 % separating gel was casted, as detailed

below:

Preparation of separating gel

Separating gel (12 %) was prepared by mixing the following reagents in the same

sequence as given in the table below,

All the above contents were mixed by swirling gently in the container and poured

into the gel casting unit using a an auto-pipette till 1.5 cm from the top edge of the plate

and over-laid with 200 L butylated water (as detailed in table-19 of appendix). After 45

minutes incubation at room temperature, butylated water was decanted and the separating

gel was rinsed with Milli-Q water.

Preparation of stacking gel

Stacking gel (4 %) was prepared by mixing the following reagents in the same

sequence as given in the table below.

Reagents (Per one gel)

1. Water 6.17 mL

2. Acrylamide mix (30 %) 8.0 mL

3. 1.5M Tris Cl pH:8.8 5.0 mL

4. 20 % SDS 100 L

5. Ammonium per-sulfate (10 % w/v) 200 L

6. TEMED 8 L

Reagents (Per one gel)

1. WFI 1.8 mL

2 Acrylamide (30 %) 0.6 mL

3 0.5M Tris Cl pH:6.8 2.5 mL

4 20 % SDS 25 L

5 Ammonium per sulfate (10 % w/v) 50 L

6 TEMED 5 L

Prepared mix was poured on the stacking gel and incubated for 45 minutes at

room temperature. Comb was removed to make wells for sample loading.

Sample loading

All the samples were heat treated in boiling water bath for 2 min, followed by

centrifugation at 3000 rpm for 2 minutes and loaded on to the gel.

Reference material, control and test samples were subjected to electrophoretic

analysis along with molecular weight markers

The electrophoretic separation of proteins was achieved at a constant voltage: 130

V with a maximum current of 200 mA. Bromophenol blue was used as a tracking dye

and the samples were allowed to run until dye front reached the bottom of the gel (~ 3.5

h).

Silver Staining of Gel

After the electrophoretic run, gel was carefully transferred to a tray containing

100 mL fixing solution (as detailed in table-22 of appendix) and incubated on a

horizontal shaker for 30 min. Later, gel was incubated with 5 % methanol (as detailed in

table-22 of appendix) for 15 min. The gel was washed thrice for 5 minutes with 100 mL

WFI. The gel was subsequently incubated in 125 mL of sodium thio-sulphate solution

(as detailed in table-22 of appendix) which was followed by three WFI washes for 30

seconds. Further, gel was incubated in 125 mL of silver nitrate solution (as detailed in

table-22 of appendix) for 25 minutes followed by two WFI washes for 1 minute each.

Later, gel was incubated in 125 mL developing solution (as detailed in table-22 of

appendix) for 10 minutes and further color development was arrested by adding 100 mL

of 5 % glacial acetic acid solution.

Gel documentation:

Stained gel image was recorded by using a gel scanner (Biorad gel doc 1000,

Mumbai, India).

N-Terminal Sequencing

N-terminal amino acid sequencing is performed by Edman degradation to

establish identity of the rhEPO protein as per method described in the European

Pharmacopoeia.

Principle:

In this method, the N-terminal-amino group is coupled with phenyl

isothiocyanate, enabling the selective cleavage of the first peptide bond, followed by

chemical conversion of the first residue into a stable amino acid residue and analysis by

RP-HPLC. Each amino acid has a characteristic retention time in the RP-

chromatography, enabling the identification of the residue by comparing it with a

reference standard. This procedure is repeated for a desired number of cycles. The

method confirms the primary sequence of the protein.

Glass plates (160 x 160 mm), spacer (1-mm thick with 6 well comb), were used


below:

Casting the separating gel:

Reagents For 7 mL (per 1gel)

1. Water 2.16 mL

2. Acrylamide mix 2.8 mL

3. 1.5M Tris-Cl pH:8.8 1.75 mL

4. 20 % SDS 35 L

5.10 % w/v Ammonium Per

sulfate (APS)

70 L

6. TEMED 3 L

All the above contents were gently mixed by swirling in a container (50 mL

falcon) and poured ~ 5 mL of solution into the casting unit.

Butylated water (500 L) (as detailed in table-19 of appendix) was gently

overlayed on the top of separation gel and allowed it for polymerization at

room temperature (RT) for 45 minutes.

Casting the Stacking gel: 4 %

Butylated water was decanted and rinsed the separating gel with Milli-Q

water.

Traces of water were removed above the separating gel using Whatman paper

and placed the comb (1 mm) between the gel plates. Stacking mix was

prepared by adding the reagents in the sequence given in the table below.

Reagents For 5 ml (for 1gel) 1. WFI 1.8 ml

2. Acrylamide (30 %) 0.6 ml

3. 0.5 M Tris HCl, pH 6.8 2.5 ml

4. 20 % SDS 25 l

5. Ammonium Per sulfate (10 % w/v)

(APS)

50 l

6. TEMED 5 l

All the contents were mixed swirling gently in a container (15 mL falcon) and

poured the stacking mix over the separating gel using a pipette followed by

allowing it to polymerize at RT for 45 min.

Sample preparation and loading

Test sample (corresponding to 10 µg protein) was withdrawn in to a 0.6 mL tube

and 6 µL of 5 X non-reducing sample buffer (as detailed in table-21 of appendix) was

added. Volume was made up to 30 µL with WFI.



and the samples were allowed to run until dye front reached the bottom of the gel (~ 1.5

h).

Electro-Transfer

Running unit was dissembled gently and marked the orientation of loading in

gel by cutting the right hand lower edge of the gel.

Gel was placed (10-15 mins) in ~15 mL of chilled transfer buffer (as detailed

in table-28 of appendix) for equilibration.

PVDF membrane was cut with the same dimensions as the gel and

equilibrated in transfer buffer for at least 15 mins.

Nylon pads were also soaked in transfer buffer and placed on pad of the

cathode (black) plate.

Five sheets of Whatman No.3 filter paper, soaked in the transfer buffer were

cut to the size of the gel and placed them on the nylon pad placed on cathode

plate (black) of Biorad-submerged system.

Equilibrated gel was carefully placed on the Whatman filter papers followed

by placing the equilibrated membrane onto the gel.

A clean glass rod dipped in transfer buffer was rolled on top of the gel to get

rid of any air bubbles trapped between the gel and the membrane.

Five sheets of Whatman No.3 filter paper soaked in ice-cold transfer buffer

were placed over the gel and once again the glass rod was rolled to remove

any air bubbles.

The cassette was closed and placed in the running unit in such a way that the

black plate of cassette faces the cathode (Black side) while white plate faces

the anode (Red side).

Electrodes were connected with the power pack and the voltage was set at

100 V for 1 hour in order to facilitate the transfer.

After the transfer got over, unit was disassembled and the membrane was

gently removed followed by staining the gel with Coomassie stain (as detailed

in table-46 of appendix).

After 5 -10 minutes, gel was transferred to a destaining solution (as detailed

in table-47 of appendix) till the bands were visible.

Based on the band size, the membrane was cut (as fine as possible) and

distained further with destaining solution.

The cut pieces of blotted membrane were, transferred to sterile, dry micro

tubes and sealed with Para film and then subjected to sequencing for first 15

amino acid analysis.

The sequence obtained after the analysis was compared with the reference

sequence given in the EPO monograph (European Pharmacopoeia)

Peptide Mapping

Peptide mapping is generally performed to establish the identity of rhEPO in

terms of its primary sequence as per the method described in the EPO monograph

(European Pharmacopoeia).

Principle:

Peptide mapping involves chemical or enzymatic digestion of proteins,

generating peptides of various lengths and hydrophobicities, which are then resolved on

reverse phase chromatography. The separation profile thus obtained depends on primary

sequence of a protein, and is considered to be its ‘fingerprint’. It is a powerful tool

capable of identifying almost any single amino acid change, resulting from events such

as point mutations and hence it is used as a test of identity. It is a comparative method

where the chromatographic profile of the protein is matched with a reference standard.

Tryptic digest of rhEPO results in well separated peptides and few clusters of

peptides when resolved on RP-HPLC. This chromatographic profile is compared with

reference standard, EPREX.

Sample Preparation

Test sample and standard were diluted using Tris-acetate buffer, pH 8.5 (as

detailed in table 75 of appendix) to obtain a concentration of 1 mg/mL solution. For

digestion 250 L of sample was used.

Procedure

Digestion

In two different microtubes, 250 L (1 mg/mL concentration) of test and

reference sample was taken. 5 L of 1 mg/mL Trypsin solution (as detailed in

table-30 of appendix) was gently added to both the tubes.

The reaction mix was incubated in a water bath at 37C for 18 h.

After 18 h, reaction was stopped by freezing.

Injection and Resolution

Test sample and reference standard were injected and perform the run using

the following conditions:

Flow rate: 0.75 mL/min to 1.25 mL/min

Method Stop time: 150 minutes

Mobile Phase A: 0.06 % v/v solution of TFA in water.

Mobile phase B: 10 % water, 90 % Acetonitrile, and 0.06 %TFA.

Injection Volume: 50 L

Maximum pressure: 250 bars

Running temperature: 30C

Absorbance: Recorded at 214 nm

Gradient used in method

Time (min)

% B (WFI+0.06 % TFA)

% A (90% ACN+ 0.06% TFA)

Flow rate (mL/min)

0 0.0 100.0 0.750 10 0.0 100.0 0.750

125 61.0 39.0 0.750 135 83.0 17.0 1.250 145 100.0 0.0 1.250 150 0.0 100.0 1.250

Data generated was integrated using integration events given below and the

qualitative peak profile of test sample was compared with that of reference standard.

Integration parameters and events

Parameters Value

Slope sensitivity 5

Peak width 0.5

Area rejected 200

Height rejected 1

Shoulder OFF

Time

0 min Integration OFF

20 min Integration ON

105 min Integration OFF

Western blot analysis

Principle

SDS-Western blot is a method generally used to detect presence of proteins in a

tissue homogenates or a mixture of different proteins. The method employs

electrophoretic separation of proteins in gel based on their molecular weight, followed by

transfer of proteins to hydrophobic membranes and then detection of a particular protein

using antibody specific against that protein.

Transfer efficiency of proteins is increased using electro transfer. After transfer,

the exposed hydrophobic patches on the membrane are blocked by incubating the

membrane with dilute protein solutions. This avoids non-specific binding of antibodies

to the membrane. The bound proteins are then detected using antibodies raised

specifically against the protein of interest. A detection system is then employed to detect

the bound antibodies. Since the antibodies are specific towards protein of interest, it is

used as a method to establish identity of test sample as rhEPO.

The method used in the present investigation is as per method described in the

European Pharmacopoeia.

Glass plates (160 x 160 mm), spacer (1-mm thick with 6 well comb), were used


below:

Casting the separating gel, (14 %)

Reagents For 7 mL (per gel)

1. Water 2.16 mL

2. Acrylamide mix 2.80 mL

3. 1.5 M Tris-Cl pH:8.8 1.75 mL

4. 20 % SDS 35 L

5. 10 % w/v Ammonium Persulfate (APS)

70 L

6. TEMED 3 L

All the above contents were mixed by swirling gently in a container (50 mL

falcon) and ~ 5 mL of solution was poured into the casting unit. Separating gel mix was

over-laid with ~500 L butylated water (as detailed in table-19 of appendix) and allowed

for polymerization for 45 minutes at RT.

Casting the Stacking gel (4 %):

Butylated water was decanted from the top of the gel and rinsed with Milli-Q

water twice.

Traces of water were gently removed using Whatman paper from the top of

separating gel and comb was placed between the gel plates.

Stacking mix was prepared by adding the reagents in the sequence given in the

table below.

Reagents

For 5 mL (per gel)

For 10 mL (per gels)

1. Water 1.8 mL 3.6 mL 2. Acrylamide (30 %) 0.6 mL 1.2 mL 3. 0.5M Tris Cl pH:6.8 2.5 mL 5.0 mL 4. 20 % SDS 25 L 50 L 5. Ammonium persulfate (10 % w/v) (APS) 50 L 100 L

6. TEMED 5L 10 L

All the above contents were mixed by swirling gently in a container (15 mL

falcon) and were poured over the separating gel using a pipette.

Gel was allowed to polymerize for at least 45 minutes.

Sample loading and gel running

All the samples were kept in the boiling water bath for 2 minutes and centrifuged

for 2-3 minutes, and later was loaded on to the gel.

Reference material (Eprex), test samples and marker subjected to electrophoretic

analysis is given below.

Samples Amount of protein loaded/well (g)

1. Test Sample 10

2. Eprex 10

3. Marker dye 10 Electro-Transfer

Running unit was dissembled gently and the orientation was marked on the

gel by cutting the right hand lower edge of the gel.

Gel was placed (10-15 min) in ~15 mL of chilled transfer buffer (as detailed

in table-28 of appendix) for equilibration.

PVDF membrane was cut with the same dimensions as the gel and was

equilibrated in transfer buffer for at least 15 min.

Nylon pads were also soaked in transfer buffer and were placed on pad of the

cathode (black) plate.

Five sheets of Whatman No.3 filter paper were soaked in the transfer buffer

which were cut to the size of the gel and placed them on the nylon pad of the

cathode plate (black) of Biorad-submerged system.

Gel that was equilibrated was carefully placed on the Whatman filter papers.

A clean glass rod dipped in transfer buffer was rolled on top of the gel to get

rid of any air bubbles trapped between the gel and the membrane.

Five sheets of Whatman No.3 filter paper that were soaked in ice-cold

transfer buffer were placed over the gel and once again the glass rod was

rolled to remove any air bubbles.

The cassette was closed and placed in the running unit in such a way that the

black plate of cassette faces the cathode (Black side) while white plate faces

the anode (Red side).

Electrodes were connected with the power pack and the following parameters

were set

Current: 400 mA

Voltage: 100 V

Run time: 1 h.

After the run, membrane was transferred to the blocking solution (as detailed

in table-35 of appendix) and incubated for 1 h with gentle shaking at RT

Development of Membrane

Blocking buffer was discarded and 25 mL of Rabbit anti rhEPO antibody

(primary antibody) solution (as detailed in table-37 of appendix) was added

followed by incubation for 1 h with gentle agitation at RT.

Primary antibody solution was decanted completely into a tube and preserved the

solution by adding 0.005 % sodium azide (as detailed in table-36 of appendix).

Membrane was washed with 1 X TBS (as detailed in table-32 of appendix) at

room temperature with gentle agitation for 15 mins.

TBS was completely discarded and 25 ml of Goat anti-rabbit ALP conjugated

antibody (secondary antibody) solution (as detailed in table-38 of appendix) was

added followed by incubation for 1 h with gentle shaking at RT.

Secondary antibody solution was decanted and preserved it by adding 0.005 %

sodium azide.

Membrane was washed with 1 X TBS at room temperature with gentle agitation

for 15 min.

Substrate solution (5 mL) BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-

Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride) were added for color

development.

When both the bands corresponding the test sample and reference were visible

completely, and are sufficiently intense, reaction was stopped by pouring off the

substrate solution and washing it with water before the color of the background

appear dark.

Iso-electric focusing (IEF)

Iso-electric focusing is intended to be used as a test of identity for rhEPO. The

method aims to compare the isoform distribution in the test sample with that of the

reference standard.

Principle

Isoforms, which have same molecular weight but differ from each other in terms

of their isoelectric point (pI), are successfully resolved using IEF. All the isoforms of

EPO are resolved using this method under denaturing condition. The distribution of

isoforms in a commercial reference preparation is used as a reference test of identity

(European Pharmacopoeia). The Coomassie stain gives a percent gradation of isoforms,

which might not be evident in highly sensitive detection systems like immunoblotting or

silver staining, due to saturation at very low amounts of protein.

Sample Loading and gel running

Following, reference material (Eprex), test samples and marker were subjected to

electrophoretic analysis along with molecular weight markers.

S. No. Samples Amount of protein loaded/well (g)

1 Test Sample 15

2 Eprex 15

3 Marker 2



and the samples were run until dye front reached the bottom of the gel (~ 1.5 h).

Coomassie Staining

Gel was transferred into the staining tray containing 10 % TCA (as detailed in

table-44 of appendix) and gently shaked for 10 min.

Gel was later transferred to 1 % TCA (as detailed in table-45 of appendix) for a

minimum of 2 h to remove the ampholytes.

Gel was rinsed in water and stained with Commassie blue R-250 (as detailed in

table-46 of appendix) for 10-20 minutes with gentle shaking.

Gel was rinsed with water to remove excess stain and the gel was destained till

bands were visible against a clear background.

Sialic acid Estimation

Principle

Sialic acid is a generic term for the N- or O- substituted derivatives of neuraminic

acid, a nine-carbon monosaccharide. Most common member of this group is N-

acetylneuraminic acid (Neu5Ac or NANA), where the amino group bears an acetyl

group. Forms of neuraminic acid where the amino group bears a glycolyl group is called

N-glycolyl neuraminic acid (NGNA).

Erythropoietin is a sialoglycoprotein which bears sialic acid residues at the ends

of sugar chains. Sialic acid is critical to metabolic activity of erythropoietin, and

increases its circulatory half life (Takeuchi and kobata, 1992), which influences the

potency of the protein. De-sialyated EPO shows no biological activity. Since sialic acid

directly affects invivo activity of EPO, its quantification is very critical.

Erythropoietin has 3 N-linked and 1 O-linked carbohydrate chains. Each of the

N-linked chains can contain a maximum of four sialic acid residues, and the O-linked

chain can contain up to two sialic acid residues. The erythropoietin molecule therefore

may contain anywhere from 4-14 sialic acid residues. As per European Pharmacopoeia

specifications, each EPO molecule should have at least 10 molecules of sialic acid.

Estimation of sialic acid is thus very critical.

The terminal sialic acid bond is very labile and is easily disrupted under mild

acidic conditions. This sialic acid then reacts with resorcinol, and forms an EPO-

resorcinol adduct which is extracted into an organic solvent and the absorbance of which

is measured at 580 nm.

Sample preparation

Different dilutions of sialic acid were prepared in the range of 2-10 µg/mL

namely, 2, 4, 6, 8 & 10 µg respectively and a standard curve was generated.

Test sample and reference standard were diluted to a concentration of 0.3

mg/mL.

Sialic acid Analysis

The test was carried out in triplicates with 100 µL of the test protein, Eprex

and sialic acid standards taken into 10 mL glass tubes.

To each tube, 1.0 mL of resorcinol reagent was added and closed with the

stopper.

After incubating in a boiling water bath for 30 min, tubes were quickly

cooled on ice.

To each tube 2.0 ml of organic solvent was added and vortexed for 5

minutes.

Contents from each tube were transferred to centrifuge tubes and

centrifuged at 8500 rpm for 10 min at 4C to separate the two phases.

Upper phase was carefully removed and the absorbance of all samples was

measured at 580 nm.

A standard curve was plotted with sialic acid concentration on x-axis and

average absorbance on y-axis.

Absorbance from triplicates of test sample, Eprex was noted.

The amount of sialic acid present in test sample and Eprex was determined

from the sialic acid standard curve using straight line equation, y=mx+c.

Sialic acids per mole of Erythropoietin was measured assuming that the

relative molecular mass of EPO as 30600 and that the relative molecular

mass of Sialic acid as 309 Average moles of Sialic acid / mole of EPO for

both test sample and Eprex were calculated using the formulae mentioned

below.

Formulae used for the calculation

Amount of sialic acid (in µg) = (Average absorbance at 580 nm –

Intercept)/ Slope

n moles of sialic acid = (µg of sialic acid in test solution A) 100

(Molecular weight of sialic acid: 309)

n moles of EPO in sample = ng of EPO in sample i.e., 30000

corresponding to 0.3 mg.

Moles of sialic acid/mole of EPO = n moles of sialic acid / n moles of

EPO.

In vitro Bioassay

A bioassay or biological assay is a biological testing system for estimating the

concentration of a pharmaceutically active substance in a formulated product or bulk

material. Through in vitro bioassay, detailed information on the biological activity of a

substance is achieved in contrast to the common physical or chemical methods. In vitro

biological assays (bioassays) have become more important to an effective quality control

program in the development of biopharmaceuticals and its manufacture over the last

decade. Various detection systems are available (Goldwasser and Sherwood, 1981;

Lappin et al., 1988) for the measurement of cell response.

The general approach of most bioassays is to perform a dilution assay, which

measures the biological responses at several doses. The principle assumption of a

dilution assay is that the active component follows the same principle of activity in both,

reference standard and sample preparation and in such case the unknown preparation

may in theory be derived by diluting with inert components or by concentrating the bulk

solution. This concept of bio-similarity may be checked with the help of the parallel-line

model (parallel-line assay), which is based on dose–response principle

(http://www.bioassay.de/index.php?id=parallel_line_method&L=zhjbsfwpmgbn).

Parallel-Line Method

Biological assays are frequently analyzed with the help of the parallel-line

method. The parallel-line model is illustrated in Figure 14. The logarithm of the doses is

indicated on the horizontal axis, whereas the corresponding responses are represented on

the vertical axis.

With the help of the parallel-line model the statistical validity of test sample in

relation to reference is based on the following assumption

(http://www.bioassay.de/index.php?id=parallel_line_method&L=zhjbsfwpmgbn),

The dose-response relationship is linear for the standard and sample preparation.

The dose-response curve has a significant slope.

The dose-response curves of the standard and sample preparation are parallel.

Figure 14: In vitro bioassay using Parallel-Line Method ( http://www.bioassay.de/index.php?id=parallel_line_method&L=zhjbsfwpmgbn)

The biological activity of proteins is defined in terms of an effective dose (ED),

such as the dose required producing 50 % of the maximum response, the ED50. However,

the ED50 tends to differ, depending upon the type of the assay systems (Robinson, 1997).

The available International Standards are intended for the calibration of local and

national standards. Few International organizations such as NIBSC (National Institute for

Biological Standards and Control), a World Health Organization (WHO) International

Laboratory for biological standards, is currently engaged in a program to develop WHO

International Standards for growth factors and cytokines of therapeutic or diagnostic

potential (Robinson, 1997). These International Standards (IS) are preparations of

growth factor or cytokine that are ampouled as per the WHO guidelines and assessed in

an international collaborative study for their suitability to serve as a standard (Storring

and Gaines Das, 1992).

The International Standards are used to define the potency in terms of (i)

International Units (IU) of therapeutic products (ii) to calibrate diagnostic immunoassays

(Vadlamudi et al., 1991) and (iii) to calibrate bioassays and immunoassays used for

biological and medical research (Robinson, 1997).

Analysis of rhEPO

Biological activity of all samples was calibrated against WHO international

standard (NIBSC) using parallel line model (Campbell, 1974). In addition, another

reference standard, Eprex (Wei et al., 2007) (commercially available reference material),

was also analyzed as an additional control. The assay is accepted to be valid if the

biological activity obtained for Eprex was within the limits (1x 105 IU/mg) proposed by

the manufacturer.

Principle:

Biological activity of rhEPO is determined based on dose dependent stimulation

of proliferation of TF-1 cells, human erythroid cells. These cells are derived from a

patient with erythroleukemia. These cells have receptors for EPO, GM-CSF and IL-3

(Valent et al., 1989) and can respond to any of the three growth factors, which made

them suitable for studying dose dependant response of EPO. Efficiency of signaling

through cytokine receptors depends critically on receptor orientation (Rashid et al.,

1998). Binding of EPO to the receptor expressed on the surface of the cell, stimulate the

internal signaling and proliferates (Kitamura et al., 2005 and Klingmuller et al., 1997).

The stimulatory effect of EPO/proliferation of cells is measured by reduction of yellow

tetrazolium MTT by metabolically active cells (this process requires active

mitochondria). The resulting intracellular purple formazone formed is solubilized and

absorbance recorded at 570 nm using a micro-plate reader (Hammerling et al., 1996).

Revival of TF-1 cells

One vial from WCB was revived into 6 mL of fresh growth medium (as detailed

in table-10 of appendix) and centrifuged at 1500 rpm for 8 minutes. Pellet was gently re-

suspended into 15 mL of fresh growth medium and contents were transferred to a T-75

cm2 flask. An aliquot of 500 µL volume was withdrawn for cell count. The flask was

incubated in an incubator at 37oC in 5 % CO2 atmosphere.

Propagation

After 72 hours of incubation, cell suspension from the flask was equally aliquoted

into three fresh T-75 cm2 flasks containing fresh TF-1 growth medium (25 mL/flask).

Assay Procedure

Day-1: Starvation

Cells from the 3 flasks were pooled and an aliquot of 500 µL was aliquoted for

cell count. The cell suspension was centrifuged at 1500 rpm for 8 minutes and the pellet

was re-suspended in assay media (as detailed in table-11 of appendix) for washing. The

total procedure was repeated thrice and finally the pellet was re-suspended in 25 mL

assay media. An aliquot of 500 µL was used for cell count and based on the count

obtained, the cell suspension was diluted to 25 104 cells/ mL, which were sufficient for

5 micro-titer plates (total: 50 mL). Cells were seeded into two fresh T-75 cm2 flasks

containing assay medium and incubated in the incubator at 37oC in 5 % CO2 atmosphere.

Remaining suspension was seeded back into the same T-75 cm2 flasks with fresh TF-1

growth medium and kept at 37oC in 5 % CO2 atmosphere as a backup.

Day-2: Protein dilution

Required volume of assay media was aliquoted into reagent reservoir. Reference

Standard (NIBSC), control standard (Eprex), control sample and test samples were

diluted to 25 IU/ mL (1 mg protein corresponds to 1 105 IU (European

Pharmacopoeia) and 100 µL was dispensed into respective wells of first row (A1 to A9)

of micro-well plate. Remaining wells of reference standard, control standard, control

sample, test sample, cell control were dispensed with 75 µL assay medium using 200 µL

multi-channel pipette. Assay medium (175 µL) was added into blank wells as shown in

the plate pattern below (Figure 15 & 16).

Four fold dilutions were carried out by transferring 25 µL serially from A1-A9 to

H1-H9 wells using 50 µL multi-channel pipettes. A volume of 50 µL was discarded after

the last dilution followed by addition of 100 µL starved cell suspension to all the wells

except blank. Plates were incubated in an incubator at 37oC in 5 % CO2 atmosphere.

Day-4: Quantification

After 48 hours of incubation, 20 µL of 0.5 % MTT (as detailed in table-2 of

appendix) was added to all the wells using repeat pipette with 1250 µL syringe. Plate

was later incubated at 37oC in 5 % CO2 atmosphere for 4 hours followed by addition of

100 µL of SDS (25 % w/v) solution (as detailed in table-3 of appendix) and incubation

over night at 25oC in an incubator in dark.

Day-5: Absorbance

After incubation, absorbance of the micro-titer plate was recorded against blank

at 570 nm using a micro-plate reader. Raw data was exported to excel sheet and graph

was plotted with log concentration on x-axis and absorbance values on y-axis. Linearity

and parallelism of samples were adjusted with respect to NIBSC (reference standard) and

the biological activity of control standard, control sample and test samples were

calculated from this plot.

NIBSC Control Sample Test Sample E CC BL

BL- Media blank

CC- Cell Control (cells with out protein)

E- Empty wells

NIBSC (Reference Standard)

Control sample (Sample obtained from Adherent cells)

Test sample (Sample obtained from adapted suspension cells)

Figure 15: Microtiter plate design to determine the potency of the test sample in

comparison with control sample and reference standard.

1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H

NIBSC Eprex Eprex E CC BL

BL- Media blank

CC- Cell Control (cells with out protein)

E- Empty wells

NIBSC (Reference Standard)

Control standard (commercial standard)

Figure 16: Microtiter plate design to determine the potency of the control standard in

comparison with reference standard.

1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H

Calculation of specific activity

Linear and parallel graph was plotted with absorbance values on y- axis versus

the log concentration (IU/mL) on x-axis for reference standard and samples. By taking

average of absorbance values of reference the mid-point on the graph was obtained.

Corresponding to this mid-point concentration value of samples was obtained using

straight-line equation. Log was converted to antilog to get concentration in IU/mL.

Finally specific activity of sample was calculated by taking ratio of concentration

(IU/mL) of reference to concentration (IU/mL) of sample and multiplied by assumed

specific activity.

Formula used in the calculations

1. Geometric mean (y) of absorbance values of reference standard was taken using

excel formula “= GEOMEAN (Y values)”

2. Slope value (m) for reference standard and sample was calculated using excel

formula “=SLOPE (y values, ,x values)”

3. Intercept value (c) for reference standard and sample was calculated using excel

formula “= INTERCEPT (y values, x values)”

4. “x” for reference standard and samples was calculated using equation for straight

line, y = mx+c i.e.. x = (y-c) / m, where, y is the geometric mean of absorbance

values of reference standard, c is intercept and m is the slope.

5. Antilog of “x” was taken to get actual x i.e. “IU/mL” value using excel formula

“=10 ^X value”

6. Specific activity (IU/mg) was calculated by the following equation,

X (IU/mL) reference standard

× X (IU/mL) sample

Note: Pre-dilution of Samples and standards were carried out based on the assumed

pecific activity 1 105 IU/mg protein (European Pharmacopoeia)

Assumed specific activity

Documents

Sreedhar Reddy PhD Thesis soft copy - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8309/9/09_chapter 4.pdf · An aliquot of the cells was seeded back into conical flak with