Analytical Method Development and Validation at PT Kalbio Global Medika: pH and Subvisible Particle Relationship Study of Ezelin (Insulin Glargine

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PREFACE

This internship report is written in accordance with the academic requirements for Internship/Field Practice (PHA4191) and is the product of the internship conducted at PT Kalbio Global Medika (KGM) from 1st July - 31st August 2022. The author was positioned as an internee in the Analytical Method Development (AnDev) division of the Research and Development (RnD) department at the company. This report regarding the subvisible particle study of a biologic product was made earnestly, but still with some limitations. Nevertheless, it is hoped that the readers may gain new insights and useful knowledge from this report.

First of all, I would like to give thanks to the Lord Jesus for His every blessing and guidance given unto me throughout my life, including my internship period. It is Him that has allowed me to achieve all things I have, and has given me the strength to reach where I stand right now. I also owe my biggest thank you to Mr. Hanslibrery S., the manager of research and development department;

Ms. Anggitaning K., my field supervisor during the internship; and all my senior scientists from the AnDev team who have welcomed me warmly and guided me at work.

I would like to give a great appreciation to my superior from i3L, Mr. Fandi S., who has given me his time, patient guidance, and thorough advice in the making of this report. Lastly, to my friends whom I am deeply indebted to: Janice Evita S., Putri Avanny J., Giovanni Anggasta, and Tasya A. Rizal, I would like to express my sincere gratitude for the support given to me throughout those two months; without them, my stay in Cikarang would've been far from enjoyable.

Jakarta, 6 October 2022

Celine Chelovna

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ACKNOWLEDGEMENT PAGE

Analytical Method Development and Validation at PT Kalbio Global Medika: pH and Subvisible Particle Relationship Study of Ezelin (Insulin Glargine)

AUTHOR:

Celine Chelovna 20010005 PHARMACY

ACKNOWLEDGED BY:

FIELD SUPERVISOR SUPERVISOR AT I3L

Anggitaning Kinanti apt. Fandi Sutanto, Ph.D

HEAD OF DEPARTMENT AT I3L

apt. Audrey Amira Crystalia, M.S

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TABLE OF CONTENTS

PREFACE 1

TABLE OF CONTENTS 3

LIST OF FIGURES 5

LIST OF TABLES 6

LIST OF ABBREVIATIONS 7

SUMMARY/ABSTRACT (MAX 1 PAGE) 8

CHAPTER 1: INTRODUCTION (MAX 3 PAGE) 1

Host Institution/Company 1

Description about the company 1

Description of department 1

Product of the Host Institution / Company 1

CHAPTER 2: PROJECT DESCRIPTION (MAX 7 PAGE) 3

Internship Program 3

Project Background 3

Objectives / Aims 3

Problem formulation and Proposed Solutions (incl. Material and Methods) 6

CHAPTER 3: FINDINGS (MAX 15 PAGE) 8

Result 8

pH Testing

Subvisible particle testing 10

Discussion 11

CHAPTER 4: CONCLUSION AND RECOMMENDATIONS (MAX 1 PAGE) 11

CHAPTER 5: SELF REFLECTION (MAX 1 PAGE) 12

APPENDICES 13

REFERENCES 14

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LIST OF FIGURES

Figure 1. Logo of PT Kalbio Global Medika

Figure 2. PT Kalbio Global Medika facility in Cikarang Figure 3. Ezelin Insulin Glargine

Figure 4. Lighthouse LS-20v.1 Liquid Sampler at Kalbio Figure 5. Group picture with some of the RnD scientists

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LIST OF TABLES

Table 1. Sample List Testing list of Ezelin Insulin Glargine Table 2. Results of sub-visible particle study

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LIST OF ABBREVIATIONS

AnDev Analytical Method Development API Active Pharmaceutical Ingredient BPOM Badan Pengawas Obat dan Makanan BSC Biosafety Cabinet

CDMO Contract Development and Manufacturing Organization CMO Contract Manufacturing Organization

DP Drug Product

DS Drug Substance

EMA European Medicinal Agency GMP Good Manufacturing Practice

HR Human Resources

LOD Limit of Detection LOQ Limit of Quantification

NMT Not More Than

KgBio Kalbe-Genexine Biologics KGM Kalbio Global Medika

OTC Over the Counter

PD Product Development

PIC/S Pharmaceutical Inspection Convention and The Pharmaceutical Inspection Cooperation Scheme

QA Quality Assurance

QC Quality Control

RnD Research and Development SOP Standard Operating Procedure

TS Technical Support

USP United States Pharmacopeia

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SUMMARY/ABSTRACT

PT Kalbio Global Medika (KGM) is a subsidiary company of PT Kalbe Farma Tbk. which is a pioneer biopharmaceutical-manufacturing company in the country. The analytical method development (AnDev) team in the research and development (RnD) department of KGM is responsible for verifications of the analytical methods used throughout the manufacturing process in KGM and for analytical studies in the case of an issue in the product manufacturing. The analytical study discussed in this report is regarding the relationship between pH and subvisible particles of Ezelin (Insulin Glargine), one of the products currently manufactured by KGM. Particulate matter in biotherapeutic products could occur due to aggregations in which based on the source could be categorized into inherent, intrinsic, and extrinsic aggregates. Formation of these aggregates are influenced by several factors, of which changes in pH level is included. The study was conducted by dividing Ezelin samples into different treatment groups where each group is adjusted to a certain level of pH using – solutions. Subvisible particle analysis was conducted using the light obscuration method using the Lighthouse LS-20v.1 Liquid Sampler. The result of the study showed that the subvisible particle count of Ezeline was neither directly nor inversely proportional to the decrease or increase of pH. It is suggested that the varying value of subvisible particle count was caused by a factor other than pH, such as contamination from personnels, the difference of pH adjustments to different treatment groups, and formation of air bubbles during mixing of the samples which may affect the light obscuration test results.

Keywords: Research and development, analytical method development, insulin glargine injection, subvisible particle test, light obscuration test

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CHAPTER 1: INTRODUCTION

1.1. Host Institution/Company

1.1.1. Description about the company

PT Kalbe Farma Tbk. is a pharmaceutical company founded in 1966 and is a dominating integrated healthcare provider in Indonesia, in which their four major work-divisions consist of the prescription drugs division (23%), the health products division (17%), the nutrition division (30%), and the distribution and logistics division (30%). These four divisions maintain a comprehensive portfolio of over the counter (OTC) and prescription drugs, nutrition products and energy drinks, and a distribution business that reach more than one million outlets throughout Indonesia.

The company has now become the biggest public healthcare company in East Asia, with a capitalization value of Rp 79,2 trillions and a sales of Rp 20,2 trillions at the end of 2017 (Kalbe, 2022) .

Figure 1.Logo of PT Kalbio Global Medika

Figure 2.PT Kalbio Global Medika facility in Cikarang

PT Kalbio Global Medika (KGM) is a pioneer

biopharmaceutical-manufacturing company in the country and is part of the PT Kalbe-Genexine Biologics (KGBio) group, which is a daughter company of PT Kalbe Farma Tbk. The company was established in 2014 and inaugurated in 2018 by Ir. Joko Widodo, the President of the Republic of Indonesia. This contract company encompasses the vision of becoming the best CMO and CDMO in the region which is driven by quality, innovation, and operational excellence; their mission is to improve health for a better life by providing high-quality and accessible biopharmaceutical products. Their vision and mission are supported by the state-of-the-art facilities and compliance to PIC/s, GMP certification by BPOM, and is EMA Qualified Person Audited.

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1.1.2. Description of department

The KGM organization is led by the Director and consists of several departments, including, but not limited to the production, human resources (HR), quality control (QC), quality assurance (QA), and RnD departments. The RnD department of KGM is led by Julianto Setiady, B.E., M.Eng., Ph.D. as RnD Head Biopharma and managed by Hanslibrery S., S.Si as RnD Manager. There are three divisions in the RnD department, which are the product development (PD), technical support (TS), and analytical method development (AnDev). As KGM is a contract company, the products manufactured by the plant are mostly orders from clients which have included their own methods of production and drug formulations along with the order. Therefore, the scope of work of the RnD department focuses mostly on optimization and verifications of the existing methods and information given by the client. Some of the work performed include process characterization, improvements on formulation of the drug, development and optimization of the production process, and development of the analytical methods required in the product manufacturing. This report will discuss the scope of work of the AnDev division, where the author was placed in for her internship program.

1.1.3. Product of the Host Institution / Company

KGM currently manufactures several technology-transfer biologics, including erythropoietin (Hemapo®), insulin glargine (Ezelin®), monoclonal antibody rituximab (Rituxikal®), and granulocyte-stimulating factor (Leucogen®). These four products have been commercialized, but are currently under process to be halal certified. The product that will be discussed in this report is the Ezelin® insulin glargine as the author’s study during the internship was regarding that product.

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CHAPTER 2: PROJECT DESCRIPTION

2.1. Internship Program

2.1.1. Project Background

The scope of general analytical chemistry includes identification (qualitative) and quantification of certain components. The technique in which an analysis is performed on a sample, whether qualitative or quantitative, is called an analytical method (Rina et al., 2021). In pharmaceutical industries, analytical methods are developed to analyze whether the active pharmaceutical ingredients (API), excipients, drug substance (DS), drug product (DP), and other related components have met the acceptance criteria; the common parameters tested include the purity, characteristics, stability, potency of the drug, and identity of components—mainly the API (active pharmaceutical ingredient) (Doltade & Saudagar, 2019). Validation of an analytical method is performed to prove whether that specific method is suitable for the desired objective and in the actual situation when compared to the reference. The validation of the method is commonly evaluated in terms of precision, accuracy, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), robustness, ruggedness, and system suitability. The AnDev team of the RnD department ensures verifications of the methods used for manufacturing KGM’s products and is responsible for analytical studies in the case of an issue in the product manufacturing.

Figure 3.Ezelin Insulin Glargine

One of the products manufactured by KGM is Ezelin®insulin glargine, which is an analog of human insulin that has been used for treatment of type 1 and type 2 diabetes by maintaining a controlled blood glucose level. This recombinant human hormone binds to the insulin receptors and may exert the functions of human insulin, including lipid regulation, blood glucose control, inducement of peripheral glucose use, prevention of liver glucose formation, and energy homeostasis. Insulin glargine is injected once daily due to its long-acting property and some regimens combine the use of rapid-acting insulin to achieve an optimal glycemic control; it is also able to be used alone and/or with other oral medication(s) (Cunningham &

Freeman, 2022; KalbeMed, 2022) .

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Insulin glargine is designed to have low solubility in a neutral pH, but high solubility in acidic conditions; this is possible due to its modifications on its amino acids such as the switch of Asn to Gly at the position 21 of the A chain and 2 additional Arg residues at position 31 and 32 of the B chain. After injection to the subcutaneous layer, the solution will remain insoluble at the physiologic pH which is more acidic compared to the product pH of 4.0. The product will then form micro-sediments which slowly releases small amounts of soluble insulin over 24 hours, starting from the onset of 1.5 - 2 hours after administration (Cunningham &

Freeman, 2022).

Subvisible particles are particles with the size of <100 µm which are unable to be visually detected by the eye (USP, 2018). These subvisible particles may be proteinaceous (protein aggregates) or non-proteinaceous (e.g., glass, silicone, and metal fragments) and coming from inherent, intrinsic, or extrinsic sources, as discussed by the USP general chapter <1787> (Carpenter et al., 2015; Mary, 2022).

Inherent particles arise from aggregations of the proteins in the drug itself or from reaction between the protein and other foreign ingredients. In the case of intrinsic particles, they are sourced from ingredients associated with the manufacturing and packaging processes of the drug, in which glass particles from vials and silicone fragments from silicone oils are common examples (Bukofzer et al., 2015).

Meanwhile, materials whose sources are excluded from the formulation and fill-and-finish processes are the origins of extrinsic particles, of which the environment and personnels involved are main contributors.

The control of particulate matter in injectable protein therapeutics has been an essential matter in pharmaceutical industries due to its association with product quality. These particles have the potential ability to impact crucial properties of the product, such as potency, clinical safety, and immunogenicity (Bukofzer et al., 2015).

One of the sources of subvisible particles is protein aggregation; it is one of the pathways of protein degradation, thus its incidence may result in a reduced product efficacy (Mary, 2022). The presence of particles may also directly cause adverse effects such as thromboembolism, inflammation, and hypersensitivity reactions.

Another concerning issue is immunogenicity which may eliminate the activity of the drug or even cause death. Certain antibodies may neutralize the protein’s activity and alter its bioavailability, leading to altered or zero response to the drug. Some of these drug-induced antibodies may even cross-react with the patient’s original endogenous protein, hence worsening the patient’s condition (Carpenter et al., 2009).

2.1.2. Scope of the project

● Perform analytical study on samples of Ezelin®insulin glargine subvisible particulate matter.

● Preparation of the sample materials: extraction of Ezelin® from cartridges and pH adjustments.

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● Perform subvisible particle count analysis on Ezelin® samples using the Lighthouse LS-20v.1 Liquid Sampler according to the SOP for sub-visible particulate matter determination.

2.1.3. Objectives / Aims

To investigate the relationship between pH and subvisible particles of Ezelin®(Insulin Glargine).

2.1.4. Problem formulation and Proposed Solutions (incl. Material and Methods)

Subvisible particles have an impact on product stability and play a role in the effectiveness of insulin glargine injection; they may boost immunogenic responses in patients, thus affecting the efficacy and safety of the product (Carpenter et al., 2015). These aggregates can either be proteinaceous or non-proteinaceous, in which formation of both types are associated with several factors such as pH. The samples from three batches of Ezelin®insulin glargine were found to have a highly varied value of subvisible particulate matter; the pH of Ezelin from all PVs are similar (3.9, 3.9, and 4.0, respectively), nevertheless, it is suspected that even the slightest difference in pH influences the Subvisible particles of the product. Accordingly, it is essential to investigate the relationship between pH and Subvisible particles of Ezelin®insulin glargine.

Table 1.Sample List Testing list of Ezelin Insulin Glargine

Sample Testing Acceptance Criteria

1^stbeaker glass (pH :4.0)

1

Appearance Clear or colorless liquid with no visible particles

Subvisible Particle

Particles ≥ 10 µm: NMT 6000/container Particles ≥ 25 µm: NMT

600/container

2

Subvisible Particle

600/container

2^ndbeaker glass (pH :4.0)

1

Subvisible Particle

600/container

2

Subvisible Particle

600/container 3^rdbeaker

glass (pH :3.6) 1 Appearance Clear or colorless liquid with no visible particles

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Subvisible Particle

600/container

2

Subvisible Particle

600/container

4^thbeaker glass (pH :3.6)

1

Subvisible Particle

600/container

2

Subvisible Particle

600/container

1

Subvisible Particle

600/container

2

Subvisible Particle

600/container

1

Subvisible Particle

600/container

2

Subvisible Particle

600/container

7^thbeaker

glass (pH :4.2) 1

Subvisible Particle

600/container

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2

Subvisible Particle

600/container

1

Subvisible Particle

600/container

2

Subvisible Particle

600/container

1

Subvisible Particle

600/container

2

Subvisible Particle

600/container

1

Subvisible Particle

600/container

2

Subvisible Particle

600/container

2.1.4.1. Materials Preparation

Insulin samples to be analyzed were from the three batches with varying subvisible particle size. All experiment processes will be performed under Biosafety Cabinet (BSC) to minimize any foreign particle that may affect the analysis result. The Ezelin samples used were the ones with rejects on the outside of packaging only and without any effect on the quality of product inside. Insulin from 200 cartridges were

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extracted and transferred to a 1 L duran bottle. The mixture was then mixed using a magnetic stirrer for 5 minutes before checking for the initial pH.

2.1.4.2. pH Determination

The insulin sample in the 1 L duran bottle was measured for initial pH using a pH meter (Mettler Toledo) which had been priorly calibrated. The Ezelin®sample was then divided into 10 beakers (100mL) with 60 mL of insulin in each beaker, which were then adjusted to different pHs using HCl (10%) and NaOH (10%) solutions;

samples in two of the beakers were adjusted to a pH of 4.0 and used as the control group, then the others were adjusted to a pH of 3.6, 3.8, 4.2, and 4.4 with two beakers in each treatment group. From each beaker, the sample was then transferred into two conical tubes (50 mL) with the amount of 25 mL in each tube.

2.1.4.3. Subvisible Particle Testing

The Ezelin® samples in each conical tube were analyzed for subvisible particle count. The specifications in USP <787> and European Pharmacopoeia General Chapter 2.9.19. limits the amount of subvisible particles of≥10 µm and≥25 µm, as shown on table X, while the smaller particles (1 - 10 µm) are yet to be addressed by both pharmacopeias. The measurement of subvisible particles according to the USP <788> can be done using the light obscuration or the microscopic particle count method. This study uses the Lighthouse LS-20v.1 Liquid Sampler to quantify subvisible particles in Ezelin samples with the light obscuration method, which is the preferred method according to Singh et al. (2010). The system was set up according to the SOP for sub-visible particulate matter determination and the experiment was also performed accordingly in a grade D laboratory to minimize external contamination.

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CHAPTER 3: FINDINGS

3.1. Result

Table 2.Results of sub-visible particle study

Beaker Target pH

Actual Final pH

Initial pH check

Acceptance Criteria

Subvisible particle Particles ≥ 10 µm

Remarks

Particles ≥ 25 µm

Remarks

S1 S2 AVG S1 S2 AVG

Control N/A 3.94 3.94

1. Particles ≥ 10 µm: NMT 6000/container

2. Particles ≥ 25 µm: NMT 600/container

Confidential

PASS

Confidential

PASS

1 4 4.03 3.94 PASS PASS

2 4 3.99 3.93 PASS PASS

3 3.6 3.64 3.96 PASS PASS

4 3.6 3.59 3.93 PASS PASS

5 3.8 3.82 3.92 PASS PASS

6 3.8 3.82 3.96 PASS PASS

7 4.2 4.21 3.94 PASS PASS

8 4.2 4.25 3.94 PASS PASS

9 4.4 4.41 3.94 PASS PASS

10 4.4 4.42 3.98 PASS PASS

*S1: Sample 1, S2: Sample 2, AVG: Average

3.1.1. pH testing

The results for the pH measurements were depicted on table 2 above. The actual pH measured after the adjustment varied slightly compared to the targeted pH (4.0, 3.6, 3.8, 4.2, and 4.4), but was ensured to only have a variation of not more than ±0.05.

3.1.2. Subvisible particle testing

The results for this sub-visible particle study were presented on table 2 above, in which all samples from different treatment groups and control groups passed the acceptance criteria. The exact values of the subvisible particle count of each sample are confidential, therefore remains unstated.

3.2. Analysis/Discussion

The presence of aggregates and particulates in biotherapeutic products is one of the primary quality issues of biologics development. Besides posing a serious threat for undesired hypersensitivity responses, these aggregates are ranging in shape and sizes which cause characterization of these particulates to become a challenge (Das, 2022). Monitoring of these particulates are usually done by visual inspection and subvisible particulate counting assays, in which visual inspection detects visible particles with the size of approximately 100 μm or larger and the latter method is used to detect subvisible particles with smaller sizes down to 10 μm. These particles can originate from external sources or can be formed through aggregation of the product itself, in

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which one or more drug molecules interact to form oligomers, whether small-sized or large-sized, depending on the amount of molecules involved.

Based on the source, particulates in biologics can be classified into three types: inherent, intrinsic, and extrinsic. Inherent particles exist from the formulation of the biologic itself and are commonly accepted to a certain level. These particles may include proteinaceous aggregates from the API itself, or excipients such as human serum albumin. Aggregation of these substances may occur due to great shearing causes, but inherent aggregates do not affect effectiveness of the drug and therefore do not threaten the safety of the patient (Zerulla-Wernitz & Maier, 2019). Intrinsic particles are inactive components which will not react with the therapeutic formulation. They come from the materials that experience contact with the product, usually during packaging, or manufacturing process, such as rubber, glass, or silicone fragments. The product of reactions between the drug ingredients and components, such as oxidation or other long-term stability issues, may also be classified as intrinsic particulates. Lastly, the extrinsic particulates are those of which originate from the environment excluded from the manufacturing process, packaging system, or container-closure systems. These particles are foreign to the product formulation or primary packaging material and are unexpected to the product. This type of particulate creates a greater risk compared to the prior types due to its unknown bioburden; they may be labeled as external contaminants and may threaten sterility of the product (Zerulla-Wernitz & Maier, 2019). The common examples of extrinsic particles include human skin particles, debris, and fibers which are not associated with the known product-contact materials (Element, 2021).

In analysis for particulates in injections, the USP <788> suggested two methods which are the microscopic particle count test and the light obscuration test, where the latter is preferred in the case of subvisible particle analysis. The light obscuration test is based on the principle of automatic size determination and size-based particle quantification from the light blockage detected. This method is unsuitable for products with high turbidity and/or viscosity, but is feasible for Ezelin® insulin glargine since the product is clear and colorless liquid which is not viscous. Another downside to this method is that formulations with the tendency to form air bubbles are prone to have an exaggerated counting. In addition, some proteins may seem semi-transparent in the light path, therefore resulting in an inaccurate particulate count. Nevertheless, the light obscuration test for subvisible particles has been used worldwide for decades and numerous commercial biotherapeutic products have proven the method to be beneficial with no adverse effects (Das, 2022).

Samples from control and all treatment groups were found to have no visible particles from direct visual inspection and were all colorless liquid. The pH adjustments to each group varies in terms of the amount of HCl (10%) and NaOH (10%) solutions added in order to obtain the level of pH similar to the desired level. After pH were adjusted in all groups, the subvisible particle count of all samples were analyzed; they were found to meet the criteria for both particles sized ≥10 µm and

≥25 µm. The amount of the particles, though varying, were also found to have no link to the levels of pH as they were not directly nor indirectly proportional to the increase or decrease of pH.

The different amount of subvisible particles between each sample may occur due to external contaminants or intrinsic protein aggregation. Particles from the environment or personnels may enter the container of the samples during transfer of samples or during exposure to air along the experiment. Formation of aggregation may also happen due to several factors other than pH, including temperature, light, ionic strength, stirring, and shaking (Wang et al., 2010). In addition, during pH adjustment each sample of the same targeted pH level experienced a different number of NaOH (10%) or HCl (10%) addition, thus differing in the amount of pH shifts (increase and decrease).

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These variables influence the formation of subvisible particles in the samples and therefore may cause the varying values. In addition, during mixing or transfer of the samples between containers, air or gas bubbles formation may occur, hence resulting in a possible inaccurate particle count as previously discussed (Das, 2022).

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CHAPTER 4: CONCLUSION AND RECOMMENDATIONS

The analysis for subvisible particle count in biotherapeutic is crucial as the presence of particulate matter in injectable biologic products poses a serious risk of immunogenic responses and possible effects on the effectiveness of the drug. Possible sources of particulate matter in biotherapeutic products are classified as inherent, intrinsic, and extrinsic. Several factors influence the formation of aggregates that are counted as particulate matter, including pH. To study the relationship between pH and subvisible particles of Ezelin insulin glargine, pH adjustments were made to Ezelin samples which are grouped into different treatments and then analyzed for subvisible particle count using the light obscuration method. The results of the study showed that all samples from control and treatment groups passed the criteria of subvisible particle count for biologics injection. The varying value of the subvisible particle count is neither directly nor inversely proportional to the levels of pH. This varying value is suggested to occur either due to the different amount of NaOH (10%) or HCl (10%) additions, external contaminants from environment or personnel, or formation of air bubbles which affects the reading of the instrument. Further study regarding the relationship between said factors with subvisible particles in Ezelin insulin glargine may be necessary in order to identify the exact cause of the formation of the subvisible particles.

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CHAPTER 5: SELF REFLECTION

The author of this report finds the experience of an internship program at KGM as a source of many new insights regarding the professional work field in a biopharmaceutical industry. The sudden change in living environment, working schedules, and the people I was surrounded by was quite a drastic shift which I felt hard to adapt to in the first week. However, thanks to the welcoming scientists in the team I was placed in and my fellow interns who accompanied me during my stay at Cikarang, I was able to sort things out better by my second week. The laboratory practices I have learned from i3L also have helped me greatly in working at KGM since I am already familiarized with most of the instruments and basic techniques required in working there.

It is without doubt that I have obtained various new knowledge regarding the scientific methods and considerations that are utilized in KGM. I have also gained new insights on the importance of time management in working in the RnD department where multiple studies and projects are going on at the same time; I have learned more about efficiency in working while still being attentive to important details; my department manager, field supervisor, and senior scientists there have also shown me how good communication is key in team work. Several other things that I have become aware of from my internship include the importance of self-discipline, work-life balance, perseverance, diligence, and mindfulness—of which these attributes are what I aim to further develop for myself.

The two-months internship I had in KGM was greatly beneficial for my self-growth, both as a scientist and as a person. I am truly grateful for the opportunity I have received which has reaffirmed my determination in progressing further in the pharmaceutical field.

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APPENDICES

.

Figure 4.Lighthouse LS-20v.1 Liquid Sampler at Kalbio

Figure 5.Group picture with some of the RnD scientists

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