Managing Raw Material Variability Over

The Life-Cycle Of A Molecule

Sigma S. Mostafa, Ph.D. Director, Process Development, Upstream

KBI Biopharma, Inc.

Session : Selection of Source Materials (Biological products)

Overview

• Raw material types • Common sources of variability • Case studies • Managing raw material variability

Types of Raw Materials • Chemical Raw Material

Culture media, feed, supplements Base and acid used for pH control Purification buffers

• Physical Raw Material

Storage bags for media, feed, supplements Tubings, connectors, Disposable cell culture bags

• Biological Raw Material

Master/Working cell bank, primary virus stock Serum Chicken eggs, tobacco plants

Common Sources Of Variabilities Over Life-Cycle Of A Molecule

• Change in secondary vendors for RMs with multiple components

• Multiple manufacturing sites receiving RM from vendor’s

multiple sites (e.g. US vs. EU sites of media vendors) • Multiple lots of hydrolysates in media • In case of physical RMs, product line may be discontinued

• Multiple WCBs

Case Studies

Case Study #1 Media performance Variability

Description PD and GMP campaign for Phase I showed no problem with cell growth. When Phase II GMP started after 1 yr of no cell culture activities, little to no growth of cells.

Investigation All Phase I cell culture done with a single medium lot (no lot to lot variability data available). Poor cell growth apparent after few passages, not immediately after vial thaw. A long list of items investigated – hydrolysates (2 types), cholesterol, iron supplement, insulin, order of addition during liquid medium preparation, pH set points at different steps of medium preparation, age of WCB, etc.

Case Study #1 Media performance Variability

Root Cause The iron supplement used in the medium had a history of variable performance. Vendor had changed the iron source in their newer products but did not notify clients who used the older products of this potential problem. Investigation and resolution of the problem took 6 months and 8 FTEs from client’s 3 sites

Case Study #2 Media performance Variability

Description PD work showed no problem with cell growth. GMP campaign at a CMO site showed half peak cell density and half titer compared to PD.

Investigation For CMO work, liquid medium was transferred in bags across country. PD work was done by hydrating powder few days before experiment. Cell culture was done by a long perfusion process. Medium degradation in Stainless steel reactor was never a problem. Thorough investigation of raw materials, WCBs, seed train steps, and production process.

Case Study #2 Media performance Variability

Root Cause Loss of vitamin K during shipment of liquid medium across country. Addition of extra vitamin K at CMO site prior to start of culture fully restored culture performance. Investigation took 1 month and 6 FTE.

Other Causes for Media/Feed Variability

• Aged liquid media/feed

• Precipitation in feed concentrate - Often caused by pH/temperature interaction

• Water used for hydration of media/feed

- pH of water source affects solubility of powder

• Selective loss of components during sterile filtration - larger components are some times lost especially if 1 micron filter is used to prevent mycoplasma

Case Study #3 Disposable Bioreactor Bag Performance Variability

Description Poor cell growth in disposable bioreactor.

Investigation Cell growth tested in multiple types of disposable bioreactors. Leachable content measured.

Root Cause High leachable content in certain types of bags caused poor cell growth.

Case Study #4 Disposable Bioreactor Bag Performance Variability

Description Breakage of impellers during mixing study.

Investigation Impellers broke while mixing study was being performed at vendor suggested RPM. Investigation on going.

Case Study #5 Particulates in Buffer Bag

Description Particulates found in buffer bag

Investigation Testing of particulates indicated that origin of particulates was c-flex tubing.

Root Cause Buffers of certain characteristics (e.g. high pH/low conductivity) when interfacing with air can degrade c-flex tubing over time.

Case Study #6 Cell bank affected during transfer

Description WCB vials had poor viability upon thaw

Investigation WCB vials thawed in PD had good viability; whereas vials thawed at manufacturing had poor viability

Root Cause Vials were shipped to PD and manufacturing separately. Vials at manufacturing were compromised during shipment/handling/storage

Managing Raw Material Variabilities

• Comprehensive RM management plan

• In-depth understanding of the supply-chain of each RM

• At least one back-up supplier for each RM

• Use of platform process to minimize change of RM

• In house data on expiration dates of key RMs

• Information on RM production process differences from site to site

• During PD use RMs planned to be used during GMP

• If possible, in house media/feed formulation development

• If project is restarted after a long gap, do a demo run with raw materials before actual run.

Questions?