Objective of the investigation:

To identify the critical manufacturing process variable/s impacting the overall yield of a
biotherapeutic protein used in cancer chemotherapy.

Abstract:
As biopharmaceutical and biotechnology industry is slowly adopting towards implementing
the initiative of Quality by Design, using statistical tools for design of experiments and data
analysis is becoming very essential [1-4]. Due to very complex manufacturing processes, its
difficult to understand how various processing parameters impact the quality and purity of
biopharmaceutical drug molecules. Implementing univariate or bivariate data analysis in such
cases can often be in efficient and results in misleading conclusions [5-6]. Recently various
case studies have addressed the topic of performing multivariate analysis on data from
fermentation and cell culture processes however there are very few examples for the use of
data analysis techniques in downstream processing of therapeutic proteins [7-8]. Downstream
processing of biopharmaceutical manufacturing process is mainly focussed on removal of
various process and product related impurities associated with a target biopharmaceutical
molecule. Downstream processing of a particular product involves various processing steps
and number of processing parameters. I have selected one such case study for the study and I
am interested in identifying the critical processing parameter/s which affects the yield and
purity of selected biotherapeutic protein. This particular protein is given in cancer treatment.
Identification of critical process parameter can help in improving the process performance
and hence the yield and purity of selected therapeutic protein product which in turn can lead
to significant cost reduction of a particular product.

Case study selected for the study:

Target product: Biotherapeutic protein produced using fermentation

Steps in manufacturing process of a selected product:

Fig. 1 indicates sequence of various unit operations involved in the manufacturing of the
selected product. Whereas Table 1 enlists various processing parameters affecting each of the
unit operation. Table 1 also enlist the value for independent variables and yields in batch 01
and batch 02 respectively. I have collected the batch data for each of the process variable for
28 manufacturing batches for which I have also recorded the yield for each of this batch and
now need to go for the data analysis to identify the list of critical variables impacting the
yield of the selected product. Data analysis can help in identifying the trends about how each
of the process variables affects the yield of therapeutic protein product.

Solublization

Refolding

Ultrafiltration

Gel filtration chromatography

Multimodal chromatography

Fig.1: Sequence of the various unit operations involved in the manufacturing process of
a selected product.

Table 01:
Sequence of the steps and various independent parameters affecting overall yield of the
product.
STEPS & PARAMETERS
Batch No.
Solubilization
IB's OD (Absorbance)
Input Protein in mg (RP-HPLC)
IB Protein Purity (%)
Refolding
Refolded Protein, mg (HPLC)
Refolding Yield (%)
Refolding Buffer pH [Container-1] pH 9.0 0.2
Refolding Buffer pH [Container-2] pH 9.0 0.2
Ultrafiltration
UF Concentrate OD
UF Concentrate Volume
UF Permeate OD
UF Total Protein by OD
Gel Filtration Chromatography-I
GFCI Output Volume (mL)
GFCI Output (OD)
GFCI Yield Percentage by OD
GFC I Total Protein by OD [mg]
GFC -I Buffer pH [Container-1] pH 5.5 0.1
GFC -I Buffer pH [Container-2] pH 5.5 0.1
GFC -I Buffer pH [Container-3] pH 5.5 0.1
GFC -I Buffer Cond. [Container-1] 1.4 - 1.6 mS/cm
GFC -I Buffer Cond. [Container-2] 1.4 - 1.6 mS/cm
GFC -I Buffer Cond. [Container-3] 1.4 - 1.6 mS/cm
Multimodal Chromatography
MMC Output Volume (mL)
MMC Output (By OD)
MMC Output Protein in mg (by OD)
MMC Percentage Yield by OD
MMC Elution Buffer pH [pH 5.5 0.1]
MMC Elution Buffer Conductivity [10.5 - 11.5 mS/cm]
Overall yield (%)

Batch no. 1

Batch no. 2

0.393
11906
74.4

0.377
11824
73.9

6356
49.9
9.04
9.00

6438
56.29
9.07
9.08

0.968
4700
0.08
5290

1.008
4900
0.079
5743

5667
0.878
78.4
5786
5.52
5.5
5.5
1.58
1.59
1.568

5591
0.94
77.6
6111
5.45
5.45
5.45
1.43
1.45
1.458

1700
1.218
2408
41.6
5.51
11.19
16.8

1864
1.237
2681
43.9
5.51
11.09
19.3

Expected outcome of the investigation:

1. Identification of impact of each independent variable (process parameter) on yield of a
therapeutic protein product.
2. If possible identification of optimum value of each process parameter leading to higher
yield of a therapeutic protein product.
3. Defining the optimum value for each process parameter leading to higher yield for
therapeutic protein product.

Topic 2
Objective 02:
To identify the critical manufacturing process variable/s impacting the various unit
operations in the manufacturing of a therapeutic protein product. In the above mentioned
problem I have separated refolding yield and chromatography yield as separate outcomes of
the process. The objective 2 of the investigation will be more focussed on identifying the
parameters impacting refolding and chromatography steps separately and to check
whether there is correlation among these unit operations and how that impacts overall
process yields.

Expected outcome of the investigation:

1. Identification of impact of each independent variable (process parameter) on yield of a
therapeutic protein product.
2. If possible identification of optimum value of each process parameter leading to higher
yield of a therapeutic protein product.

Table 02: Sequence of the steps and various independent parameters affecting refolding
and chromatography yields of the therapeutic protein product.
STEPS & PARAMETERS
Batch No.
Solubilization
SIB's OD
Input Protein in mg (RP-HPLC)
IB Protein Purity (%)
Refolding
Refolded Protein, mg (HPLC)
Refolding Yield (%)
Refolding Buffer pH [Container-1] pH9.0 0.2
Refolding Buffer pH [Container-2] pH 9.0 0.2
Ultrafiltration/Concentration
UF Concentrate OD
UF Concentrate Volume
UF Permeate OD
UF Total Protein by OD
Gel Filtration Chromatography-I
GFCI Output Volume (mL)
GFCI Output (OD)
GFCI Yield Percentage by OD
GFC I Total Protein by OD [mg]
GFC -I Buffer pH [Container-1] pH 5.5 0.1
GFC -I Buffer pH [Container-2] pH 5.5 0.1
GFC -I Buffer pH [Container-3] pH 5.5 0.1
GFC -I Buffer Cond. [Container-1] 1.4 - 1.6 mS/cm
GFC -I Buffer Cond. [Container-2] 1.4 - 1.6 mS/cm
GFC -I Buffer Cond. [Container-3] 1.4 - 1.6 mS/cm
Multimodal Chromatography
MMC Ouput Volume (mL)
MMC Output (By OD)
MMC Output Protein in mg (by OD)
MMC Percentage Yield by OD
MMC Elution Buffer pH [pH 5.5 0.1]
MMC Elution Buffer Conductivity [10.5 - 11.5 mS/cm]
Overall yield (%)
Yield After Refolding (%)
Chromatography Yield (%)

Batch no. 1

Batch no. 2

0.393
11906
74.4

0.377
11824
73.9

6356
49.9
9.04
9.00

6438
56.29
9.07
9.08

0.968
4700
0.08
5290

1.008
4900
0.079
5743

5667
0.878
78.4
5786
5.52
5.5
5.5
1.58
1.59
1.568

5591
0.94
77.6
6111
5.45
5.45
5.45
1.43
1.45
1.458

1700
1.218
2408
41.6
5.51
11.19
16.8
34.5
38.3

1864
1.237
2681
43.9
5.51
11.09
19.3
39.6
48.5

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