dtr 1, 45273, cuno, 2-26-09

Round Lake, Illinois 60073COVER PAGE

REPORTStudy Number Class Type of Document

45273 B Data Transmittal Report 1

Title

Analytical Testing for Samples from CUNO 70CP Resin Changes for Albumin, IGIV and A1PI Processes

Period Covered (report) Optional Tracking Number(s)

Project Number Customer Customer’s Division

36819555 Mo Azari Bioscience

Additional Distribution (Original to Archives)

Mahmood Azari, WESTLAKE VILLAGE CA., Todd Wielgos, Chyung Cook and Jim Catarello, WG2-2S, Technology Resources Archives, WG1-1S

APPROVALSName Division Signature DateStudy DirectorJim Catarello Technology Resources

Study Director’s ManagementTodd Wielgos Technology Resources

Verifier Technology Resources

For Reports: Authorized for appropriate use outside the company in compliance with Baxter policies regarding use of BAXTER CONFIDENTIAL information? Yes, _______Initials of Study Director’s Department Head or designee and approval signature required above.

Contributing Individuals (applicable for reports)Liqiong Fang, Bev Johnson, Anthony Tomaso, Chris Cullen

The date that the last person signs this document is to be used when referencing this document.

For Protocols: Study must not begin until all approval signatures are obtained

For Reports: Report must not be issued until all approval signatures are obtained

of 18BAXTER CONFIDENTIAL

Reference Document: YY-005-003, YY-005-005 Form Number: FORM179(current version) Form Issue Date: 04-14-2003

Study Number: 45273 of 18Data Transmittal Report 1

BACKGROUND

The Baxter Bioscience division at the Los Angeles, California manufacturing facility (reference 1) is currently evaluating the impact of change in the resin of the CUNO depth filter media 70 CP grade on the efficiency and product quality at pilot scale. CUNO reports that supply of the current resin is constrained and that there will be no more supply of the current resin on January 1st, 2009. The supply of pads fabricated from the current resin is estimated to extend as short as March 31st, 2009.

The change in resin include that CUNO’s replacement resin is supplied dispersed in a liquid a liquid while the current resin is supplied as a powder. In melamine formaldehyde resins, the formaldehyde to melamine ration in the current resin has a target ratio of 2.7 while the replacement resin has a target ratio of 3.2. The replacement vendor methylates the resin to improve stability in its liquid dispersed form. The vendor further reports that these methyl groups are removed during the resin preparation process.

The structures of the current and replacement resins as provided by the vendor are shown below, showing the presence of the methyl groups (-R) which the vendor reports are released during the resin preparation (curing) process.

Figure 1: Melamine Formaldehyde Resin Structure

A diagram for the showing the location of the CUNO pads is shown below.

BAXTER CONFIDENTIAL


PURPOSE

The objective of this data transmittal report is to report the results from analytical testing on various process samples from the productions of Albumin, IGIV or A1PI (Alpha-1 Phosphatase Inhibitor) from the Baxter Bioscience facility in Los Angles, California. Size Exclusion Chromatography coupled with Multi-Angled Laser Light Scattering (SEC-MALLS) was used to evaluate product integrity and Mass Spectroscopy methods were used to evaluate all “control” and “test” resin stability samples. NMR (Nuclear Magnetic Resonance) Spectroscopy was only performed on samples were there were differences between the control and test samples.

It was determined that FTIR (Fourier Transform Infrared Resonance) was not needed for this study. However, used to evaluate resin stability.

TEST ARTICLES

Four sets of samples were received from the Baxter Bioscience Division located in Los Angeles, California. Set 1 was received on 10-17-08 and contained 23 samples. Set 2 was received 10/30/08 and contained 32 samples. Set 3 was received 11-13-08 and contained 57 samples. Set 4 was received 12-11-08 and contained 78 samples. Sample sets 1 to 3 were immediately placed in 2-8C storage upon their arrival. On 12-2-08, sample sets 1 and 2 were moved to -80C storage and on 12-6-08 set 3 was moved to -80C storage. Sample set 4 was immediately placed in -80C storage upon its arrival. All samples were logged in and given Round Lake numbers as a reference.

Control and Test samples from the Baxter Bioscience Facility in Los Angles, California that were collected during the production of Suspension B @ 17%, II+III Extract and Fr IV-4 @ 40% for the production of albumin and IGIV were received for testing. In addition, WFI and Buffer samples were also received for testing. A detailed description and sample clarification of the labeled samples received is described below:

Sample Clarification Label Clarification Proposed

Alternate Label

Starting Material (Suspension before CUNO clarification)

Raw Material sample (either II+III Extract, Fr IV-4 @ 40%, or Susp. B at 17%) from manufacturing that never came into contact with CUNO media.

No

WFI Rinse Water for Injection (WFI) that was run through the CUNO filter media. WFI is run to wet the pads.

No

Pooled Pre-Wash Solution

Buffer run through the CUNO filter media prior to filtering the protein solution. Buffer is run to equilibrate the pads and filter press to the desired temperature and pH.

Pre-filtration Wash

Pre-Wash Solution Buffer run through the CUNO filter media prior to filtering the protein solution. Buffer is run to equilibrate the pads and filter press to the desired temperature and pH. Identical to “Pooled Pre-Wash Solution”

Pre-filtration Wash

Filtrate at the End of Filtration

Sample of filtered protein solution at the end of filtration. Filtrate of the II+III extract, Fr IV-4 @ 40%, or Susp. B @ 17%.

Filtrate

Pooled Filtrate Sample after Post Wash

Sample of the wash solution used to flow through the filter media after filtering the protein solution. This ensures full recovery of the product. Post wash uses the same buffer as the Pre-Wash.

Post-Filtration Buffer Wash

Buffer Buffer Control that had never came into contact with the CUNO Media

The samples received from each sample set (1 to 4) are described the Tables 1 to 4 in the Results section of this data transmittal report.

BAXTER CONFIDENTIAL


EXPERIMENTAL DESIGN

All samples were centrifuged at either 2000 or 5000 rpm prior to analytical evaluation to separate precipitated protein. The supernatants of the centrifuged samples were submitted for analysis. The WFI and buffer samples were not centrifuged since there was no protein in those samples.

It was decided that FTIR (Fourier Transform Infrared Resonance) would not be performed. To minimize sample testing, NMR (Nuclear Magnetic Resonance) Spectroscopy and GC-MS (Gas Chromatography–Mass Spectrometry) would be only performed on samples that had chromatographic differences between the “control” and “test” samples by LC-MS or SEC.

Size Exclusion Chromatography coupled with Multi-Angled Laser Light Scattering (SEC-MALLS) with refractive index, UV at 220nm and 280nm detection was used to evaluate product integrity. LC-MS was used to evaluate resin stability and extractable material. Analytical Methods:

A. SEC-MALLS – Refractive Index (RI), UV at 220nm and 280nm Detection – PRODUCT INTEGRITY

SEC-MALLS was used to evaluate product integrity by comparing the molecular weight results for protein samples before and after filtration obtained for the control and test CUNO resin process runs. The RI, UV at 220nm and 280nm chromatographic profiles were compared for samples collected at similar time-points during the control and test sample runs. The SEC-MALLS method conditions used are described below:

Equipment

An Agilent 1100 HPLC System coupled with Wyatt Technology DAWN HELEOS, QELS (Quasi-Elastic Light Scattering and Optilab rEX differential refractive index (dRI) and UV detector was used to evaluate the test articles.

SEC-Conditions:Analytical Columns: Superdex 200.Mobile Phase: PBS Buffer, pH 7.4.Flow Rate: 0.7 mL/minute.Column Temperature: Room temperature.Sample Temperature: 5ºC.Injection Volume: 100µL.Runtime : 50 minutes.Detection : Light Scattering, Wyatt DAWN HELEOS, s/n. 263-H.

Refractive Index (RI), Wyatt Optilab rEX, s/n. 479-rEX.Quasi-Elastic Light Scattering (QELS), s/n. QH-114.UV at 220 and 280nm.

Test samples were injected (after centrifugation) directly into the SEC-MALLS system. No further dilution was performed. A BSA sample was injected for detector alignment, band broadening and normalization purposes.

B. BCA Total Protein Assay

The assays were performed according to the manufacture instructions.

C. NMR Spectroscopy

Added 10% deuterium oxide to each sample prior to analysis. Followed Technology Resources Standard Procedure PA011005, Operation and Maintenance of the Bruker Avance III 600 and Avance I 400 NMR Spectrometers. Effective 9/26/07.

BAXTER CONFIDENTIAL


D. Mass Spectroscopy

Samples were assayed directly. No further dilutions or sample treatments were made prior to analysis.

Followed Technology Resources Standard Procedure 1109105, Calibration, Operation and Maintenance of Mass Spectrometers and their Data Systems. Effective 2/17/08.

LC Conditions:

Column: Phenominex Synergi; Polar –RP

Mobile Phase: A = 25mM NH4OAc (Ammonium Acetate) / 25mM HOAc (Acetic Acid).B = AcetonitrileC = Methanol

Gradient: Time %A %B %C0 90 5 52 90 5 520 5 90 523 5 90 524 90 5 530 90 5 5

Flow Rate: 0.5ml/min.Column Temp.: 25CInj. Volume: 50LDetection: Water Q-tof Micro. Mass Spectrometer

E. GC-Mass Spectroscopy

Samples were assayed directly. No further dilutions or sample treatments were made prior to analysis.

Followed Technology Resources Standard Procedure PA009007, Operation and Maintenance of Gas Chromatographs. Effective 8/17/08. Followed Technology Resources Standard Procedure 1109105, Calibration, Operation and Maintenance of Mass Spectrometers and their Data Systems. Effective 2/17/08.

RESULTS

All samples were assayed by SEC-MALLS with RI, UV at 220nm and 280nm detection and by LC-MS. The RI, UV at 220nm and 280nm chromatographic profiles were compared for samples collected at similar time-points during the control and test sample runs. Samples showing chromatographic differences between test and control at similar time-points were submitted for NMR and GC-MS analysis. All samples were measured for protein content using the BCA method. The results for the 4 sets of samples submitted are summarized in tables 1 to 4.

BAXTER CONFIDENTIAL


Table 1: Summary of Sample Set 1 (Received 10/17/08) from LA

RL Number Sample I.D. Suspension Lot

NumberMedia Lot Number

Date Time Control or Test

BCA(mg/mL)

SEC-MALLS

MW g/mole

RI SECUV 280

SECUV 220

LC-MS NMR GC-MS

1 Starting Material (Suspension Before CUNO Clarification)

LB0820509 39214 10/7/08 11:38 CONTROL 5.387 122,600 --- --- --- --- NA NA


LB0820509 39446 10/7/08 11:38 TEST 5.282 125,600 X X X + 11M NA NA

2 Starting Material (Suspension before CUNO clarification)

LB0850512 39214 10/10/08 14:20 CONTROL 13.815 60,230 --- --- --- --- NA NA

13 Starting Material (Suspension before CUNO clarification)

LB0850512 39446 10/10/08 14:20 TEST 13.888 56,200 X X X X NA NA

14 Starting Material (Suspension before CUNO Clarification)

LB0890532 39446 10/14/08 13:12 TEST 10.484 197,700 NC NC NC NC NP NP

3 Pooled Pre-Wash Solution LB0820509 39214 10/7/08 14:04 CONTROL 0 NA --- --- --- --- NR ---15 Pooled Pre-Wash Solution LB0820509 39446 10/7/08 14:04 TEST 0 NA X - 39M X Large

20MNP X c

4 Pooled Pre-Wash Solution LB0850512 39214 10/13/08 09:21 CONTROL 0 NA --- --- --- --- NA NA16 Pooled Pre-Wash Solution LB0850512 39446 10/13/08 09:22 TEST 0 NA X X X X NA NA5 Pooled Pre-Wash Solution LB0890532 39214 10/15/08 09:12 CONTROL 0 NA --- --- --- --- --- ---17 Pooled Pre-Wash Solution LB0890532 39446 10/15/08 09:22 TEST 3.028 142,900 10 to

24M Large 18M,

39MLarge 18M,

+ 15 and 20M

X a X b

6 Pooled Filtrate Sample After Post-Wash LB0820509 39214 10/7/08 15:38 CONTROL 4.351 123,000 --- --- --- --- NA NA18 Pooled Filtrate Sample After post Wash LB0820509 39446 10/7/08 15:39 TEST 5.199 119,600 X X X + Small

11MNA NA

7 Pooled Filtrate Sample after Post Wash LB0850512 39214 10/13/08 11:49 CONTROL 14.513 53,580 --- --- --- --- NA NA19 Pooled Filtrate Sample after Post-Wash LB0850512 39446 10/13/08 11:49 TEST 14.139 51,080 X X X X NA NA8 Pooled Filtrate Sample After Post-Wash LB0890532 39214 10/15/08 11:10 CONTROL 5.811 152,600 --- --- --- --- NA NA20 Pooled Filtrate Sample After Post-Wash LB0890532, 39446 10/15/08 11:10 TEST 10.969 162,100 X X X X NA NA

9 Filtrate Sample at the End of Filtration LB0820509 39214 10/7/08 15:15 CONTROL 4.389 125,300 --- --- --- --- NA NA21 Filtrate Sample at the End of Filtrate LB0820509 39446 10/7/08 15:15 TEST 4.012 124,200 X X X X NA NA10 Filtrate Sample at the End of Filtration LB0850512 39214 10/13/08 11:29 CONTROL 13.549 52,470 --- --- --- --- NA NA22 Filtrate Sample at the End of Filtration LB0850512 39446 10/13/08 11:29 TEST 13.252 52,780 X X X X NA NA11 Filtrate Sample at the End of Filtration LB0890532 39214 10/15/08 10:35 CONTROL 8.974 151,200 --- --- --- --- NA NA23 Filtrate Sample at the end of Filtration LB0890532 39446 10/15/08 10:38 TEST 11.831 169,700 X X X X NA NA

X = Same Profile as the Control.

X c = Same GC-MS Profile as the Control. Peaks at approximately 19m = methanol, 23m = ethanol, 33.5m = 1-propanol. X a = Similar NMR spectrum as control plus resonances between 2.5 and 3.2 ppm. Sample was submitted for GC-MS analysis. X b = Similar GC-MS Profile as the Control, however, larger peaks in “Test” than “Control” at approximately 19m = Methanol, 23m = ethanol, 33.5m = 1-propanol. NA = No testing required; NP = Performed; NC = No Control for Comparison; NA = Not Applicable (No Light Scattering Response to Determine Molecular Weight).M = Peak Elution time in “Minutes”.(-) = Peak Absent(+) = Additional Peak Present.

BAXTER CONFIDENTIAL


The results in Table 1 show that the majority of samples showed no differences in the chromatographic profiles for SEC-RI, SEC-UV at 280nm, SEC-UV at 220nm or LC-MS. In addition, the molecular weight results and SEC profiles were consistent between the “test” and “control” samples indicating that the “test” CUNO resin had no impact on the products integrity. Therefore, the “test” CUNO resin had no impact on the samples produced during the production of Lots LB0820509, LB0850512and LB0890532.

There were a few differences between test and control samples. Results in Table 1 show that the profiles and results for test samples of the “Pooled Pre-Wash Solutions“ for RL numbers 15 and 17 were different than their corresponding controls (RL numbers 3 and 5).

The BCA results for RL number 17 showed the presence of protein. In addition, the SEC-RI (see figure 1), UV at 280nm (see figure 2) and 220nm profiles for “test” sample, RL number 17, were different than there corresponding control (RL number 5). The results for the UV at 280nm also confirms the presence of protein in sample the “Pooled Pre-Wash Solution“ sample for RL number 17.

Figure 1: SEC Refractive Index Overlay of Pooled Pre-Wash Solutions from Lot LB0890532.

Figure 2: UV at 280nm Overlay of Pooled Pre-Wash Solutions from Lot LB0890532.

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0-20

0

25

50

75

100

125

150

180

1 - 26544099_JC #11 [modified by catarej] 5 UV_VIS_12 - 26544099_JC #23 [modified by catarej] 17 UV_VIS_1mAU

min

21 1 - 13.008 2 - 17.949 3 - 30.3134 - 32.3325 - 33.7276 - 35.595 7 - 38.824

WVL:280 nm

Min

Ar

= 5

.00

0

Since the Pooled Pre-Wash Solution samples were collected before the protein was filtered through the CUNO resin, it was determined that RL sample 17 was most likely contaminated with protein and was not attributed t othe CUNO resin.

BAXTER CONFIDENTIAL

RL-17, Test

RL-5, Control

RL-17, Test

RL-5, Control


To look for other non-protein differences between the “control” (RL number 5) and “test” (RL number 17) samples, the samples were submitted for LC-MS analysis. The results for the LC-MS are shown in figure 3.

Figure 3: LC-MS Stacked Spectra of Pooled Pre-Wash Solutions from Lot LB0890532.

C-5

Time4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

%

3

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

%

13

FK050806 1: TOF MS ES+ TIC

3.82e422.63

11.217.697.26

2.47

8.29

8.54

8.71

17.01

FK050820 1: TOF MS ES+ TIC

1.91e520.51

18.8515.08

8.017.697.23 8.29

11.37

15.99

15.57

20.80

21.11

22.49

The LC-MS profile showed the presence of peaks in the “test” solution at approximately 15 and 20 minutes that were not present in its corresponding control. To investigate the differences seen in the LC-MS spectra, “test” sample RL number 17 and “control” sample RL number 5 was submitted for NMR analysis. The results for the NMR spectra difference are shown in figure 4.

Figure 4: NMR Stacked Spectra of Pooled Pre-Wash Solutions from Lot LB0890532.

25706-171-B, MS-17 test, 1H, Catarello, 36819555, 45273

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

25706-171-A, MS-5 control, 1H, Catarello, 36819555, 45273

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

Baxter Technology Resources, Round Lake IL 60073, AV400 RLS27611/AV600 RLS25079, SOP PA-011-005 (9/26/07)

-FOR BAXTER USE ONLY-

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Results from the NMR spectra comparison showed additional small resonances between 2.5 and 3.2ppm for test sample RL number 17 that were not seen in its corresponding control (RL number 5). The samples were then submitted for GC-MS analysis determine whether or not there was further contamination besides the additional protein observed in sample 17.

BAXTER CONFIDENTIAL

Control, RL # 5

Test, RL # 17

Control, RL # 5

Test, RL # 17

Spectral Differences


The GC-MS results for the control (RL number 5) and test (RL number 17) are shown in Figures 5a and 5b.

Figure 5a: GC-MS Overlay Profile of Pooled Pre-Wash Solutions from Lot LB0890532.

Figure 5b: GC-MS Overlay Profile of Pooled Pre-Wash Solutions from Lot LB0890532 - ENLARGED.

10.00 15.00 20.00 25.00 30.00 35.00 40.00

100000

150000

200000

250000

300000

350000

400000

450000

Time-->

Abundance

TIC: tj020209010.D\data.ms

Sample 17

Sample 5

TIC: tj020209012.D\data.ms (*)

The 1-propanol is the internal standard. The results in figures 5a and 5b show the suspected methanol along with the presence of acetamide, methyl acetate, and ethanol. Acetamide, methyl acetate, methanol and ethanol were seen in both the “test” (RL number 17) and control samples (RL number 5), however, they were in higher concentration in the test sample. There were some small “unknown peaks around 35minutes that were seen in the test that were not seen in the control. In sample 17, the peak at ~19 minutes is most likely methanol, however the spectrum has some larger ions that make it possible that it could be t-butanol.

Based on the analytical results for “test” RL sample17, the sample is contaminated with protein and has higher methanol and ethanol amounts compared to its control. Since buffer is run through the CUNO filter media prior to filtering the protein solution for the Pooled Pre-Wash Solution samples and there was no protein was passed through the CUNO resin, the contamination of protein could not be due to the CUNO resin. The higher amounts of methanol and ethanol observed in RL sample 17 compared to its control may be due to an incomplete filter wash process.

BAXTER CONFIDENTIAL

Methanol

Ethanol

1-propanol

Acetamide

Methyl Acetate

Unknowns


The other “test” sample, RL sample 15, a “Pooled Pre-Wash Solution”, showed a large 20 minute peak in its LC-MS profile that was much smaller in its corresponding “control” sample, RL sample 3 (see Figure 6).

Figure 6: LC-MS Stacked Spectra of Pooled Pre-Wash Solutions from Lot LB0820509.

This sample did not contain protein, but was sent for GC-MS analysis to investigate this large 20-minute peak in its LC-MS profile. The GC-MS profile for “test” RL sample 15, a “Pooled Pre-Wash Solution”, and its corresponding “control” sample, RL sample 3 is shown in Figure 7.

Figure 7: GC-MS Overlay Profile of Pooled Pre-Wash Solutions from Lot LB0820509.

The results in the GC-MS profile showed the same profiles for both the “test” RL sample 15 and its corresponding “control” sample, RL sample 3, therefore, the larger 20-minute peak in the LC-MS profile could not be attributed to the components identified in figure 7 and there is no significant contribution from the CUNO resin.

A small peak at approximately 11 minutes was observed in the LC-MS profile for the “test” starting material (RL number 12, Suspension Before CUNO Clarification) that was not seen in its corresponding “control” starting material (RL number 1). Since this material was starting material and had not been exposed to the CUNO resin, the small difference is not attributed to the CUNO resin material. The small 11-minute peak was seen again in the “test” Pooled Filtrate Sample After Post Wash sample (RL number 18, Lot LB0820509). This was carried over from the starting material (RL number 12, Lot LB0820509) and is not attributed to the CUNO resin material. In addition, the small 11-minute was gone at the final product, Filtrate Sample at the End of the Filtration, RL number 21.

BAXTER CONFIDENTIAL

Large 20-minute peak

Control, RL # 3

Test, RL # 15

1-propanol

acetimide

methyl acetate

methanol

ethyl acetate

ethanol


A second sample set was received 10-30-08. The results for sample set 2 are summarized in Tables 2a and 2b for Lots LB0850540, LB0850527 and LB0890558.

Table 2a: Sample Set 2 – LA Samples, Received 10-30-08 (Lots LB0850540 and LB0850527).

Number

Sample I.D. Suspension Lot Number

Media Lot Number


Total Protein

(mg/mL)

SEC-MALLS(MW = g/mole)

RI SEC-UV at 280nm

SEC-UV at 220nm

LC-MS


LB0850540 39611 10/20/08

13:49

CONTROL

12.221 58,410 --- --- --- ---


LB0850527 39602 10/16/08

15:08

CONTROL

13.476 37,650 --- --- --- ---


LB0850540 39500 10/20/08

13:49

TEST 13.158 57,930 X X X X


LB0850527 39488 10/16/08

15:08

TEST 14.055 49,930 X X X X

2 Pooled Pre-Wash Solution LB0850540 39611 10/21/08

09:29

CONTROL

0.087 NA --- --- --- ---


09:15

CONTROL

0.0675 NA --- --- --- ---


09:29

TEST 0.0495 NA X X X X


09:15

TEST 0.0525 NA X X X X

3 Pooled Filtrate Sample after Post Wash

LB0850540 39611 10/21/08

11:08

CONTROL

13.23 50,420 --- --- --- NC


LB0850537 39602 10/17/08

11:04

CONTROL

13.541 48,040 --- --- --- NC


LB0850540 39500 10/21/08

11:08

TEST 12.89 49,040 X X X NC


LB0850527 39488 10/17/08

11:04

TEST 13.478 46,390 X X X NC

4 Filtrate Sample at the End of Filtration

LB0850540 39611 10/21/08

10:50

CONTROL

12.679 51,630 --- --- --- ---


LB0850527 39602 10/17/08

10:45

CONTROL

14.584 41,890 --- --- --- ---


LB0850540 39500 10/21/08

10:50

TEST 13.975 48,580 X X X X


LB0850527 39488 10/17/08

10:45

TEST 13.150 47,080 X X X X

X = Same as Control ProfileNC = Not Compared NA = Not Applicable, No RI signal to Accurately Determine Molecular Weight.

BAXTER CONFIDENTIAL


Table 2b: Sample Set 2 – LA Samples, Received 10-30-08 (Lot LB0890558)

Number


Media Lot Number


Total Protein

(mg/mL)


RI SEC-UV at 280nm

SEC-UV at 220nm

LC-MS


LB0890558 39602 10/22/08

14:55

CONTROL

14.617 40,650,000

--- --- --- ---


LB0890558 39602 10/22/08

14:55

CONTROL

15.308 7,667,000 --- --- --- ---


LB0890558 39602 10/22/08

14:55

CONTROL

15.259 2,113,000 --- --- --- ---


LB0890558 39488 10/22/08

14:55

TEST 16.064 56,710,000

X X X X


10:28

CONTROL

0.0660 NA --- --- --- ---


10:28

CONTROL

0.0695 NA --- --- --- ---


10:28

CONTROL

0.0675 NA --- --- --- ---


10:28

CONTROL

0.0030 NA --- --- --- ---


LB0890558 39602 10/23/08

11:35

CONTROL

9.891 472,600 --- --- --- ---


LB0890558 39602 10/23/08

11:35

CONTROL

9.6595 397,100 --- --- --- ---


LB0890558 39602 10/23/08

11:35

CONTROL

10.393 543,900 --- --- --- ---


LB0890558 39488 10/23/08

11:29

TEST 9.761 565,900 X X X X


LB0890558 39602 10/23/08

11:08

CONTROL

11.506 626,200 --- --- --- ---


LB0890558 39602 10/23/08

11:08

CONTROL

11.308 594,000 --- --- --- ---


LB0890558 39602 10/23/08

11:08

CONTROL

11.521 696,600 --- --- --- ---


LB0890558 39488 10/23/08

11:05

TEST 11.563 479,500 X X X X

X = Same as Control ProfileNA = Not Applicable, No RI signal to Accurately Determine Molecular Weight.

The results in Tables 2a and 2b show that there was no differences found in the chromatographic profiles for SEC-RI, SEC-UV at 280nm, SEC-UV at 220nm or LC-MS. In addition, the molecular weight results and SEC profiles were consistent between the “test” and “control” samples indicating that the “test” CUNO resin had no impact on the products integrity. Therefore, the “test” CUNO resin had no impact on the production of Lots LB0850540, LB0850527 and LB0890558.

BAXTER CONFIDENTIAL


A third set of samples was received on 11-13-08 and 11-21-08. The results for sample set 3 are summarized in Table 3 for Lots LB0820580, LB0890585 and LB0820548.

Table 3: Set 3 – LA Samples, Received 11-13-08 and 11-21-08

RLNumber


Media Lot Number


Total Protein

(mg/mL)


RI SEC-UV at 280nm

SEC-UV at 220nm

LC-MS NMR


LB0820580 39611 11/7/08 14:35 CONTROL 5.643 128,800 --- --- --- --- NA


LB0820580 39611 11/7/08 14:35 CONTROL 5.779 128,300 --- --- --- --- NA


LB0820580 39500 11/07/08 14:35 TEST 5.732 124,700 X X X X NA


LB0820580 39500 11/7/08 14:35 TEST 5.618 133,000 X X X X NA


LB0890585 39611 10/28/08 17:03 CONTROL 15.017 213,300 --- --- --- --- NA


LB0890585 39611 10/28/08 17:03 CONTROL 15.247 336,500 --- --- --- --- NA


LB0890585 39500 10/28/08 17:03 TEST 6.587 314,200 X X X No 15.1 and 16.1 Peaks2

NA


LB0890585 39500 10/28/08 17:03 TEST 14.916 316,600 X X X X NA


LB0820548 39602 10/27/08 13:57 CONTROL 6.867 151,300 --- --- --- --- NA


LB0820548 39602 10/27/08 13:57 CONTROL 7.231 154,700 --- --- --- --- NA


LB0820548 39488 10/27/08 13:57 TEST 6.815 153,900 X X X X NA


LB0820548 39488 10/27/08 13:57 TEST 14.554 133,900 X X X Peaks at 15.1 and 16.13

NA

11 Pooled Pre-Wash Solution LB0820580 39611 11/10/08 10:05 CONTROL BDL CNBD --- --- --- --- NA12 Pooled Pre-Wash Solution LB0820580 39611 11/10/08 10:05 CONTROL BDL CNBD --- --- --- --- NA14 Pooled Pre-Wash Solution LB0820580 39500 11/10/08 10:05 TEST BDL CNBD Smaller 47m X Smaller 45m X NA19 Pooled Pre-Wash Solution LB0820580 39500 11/10/08 10:05 TEST BDL CNBD Smaller 47m X Smaller 45m X NA48 Pooled Pre-Wash Solution LB0820548 39602 10/28/08 09:28 CONTROL BDL CNBD --- --- --- --- NA49 Pooled Pre-Wash Solution LB0820548 39602 10/28/08 09:28 CONTROL BDL CNBD --- --- --- --- NA38 Pooled Pre-Wash Solution LB0820548 39488 10/28/08 09:28 TEST BDL CNBD X X X X NA46 Pooled Pre-Wash Solution LB0820548 39488 10/28/08 09:28 TEST BDL CNBD X X X X NA22 Pooled Pre-Wash Solution LB0890585 39611 10/29/08 13:30 CONTROL BDL CNBD --- --- --- --- NA30 Pooled Pre-Wash Solution LB0890585 39611 10/29/08 13:30 CONTROL BDL CNBD --- --- --- --- NA26 Pooled Pre-Wash Solution LB0890585 39500 10/29/08 13:30 TEST BDL CNBD X Small 4-10

mSmaller 45m X NA

31 Pooled Pre-Wash Solution LB0890585 39500 10/29/08 13:30 TEST BDL CNBD X No 4-10 m Smaller 45m X NA1 Pooled Pre-Wash Solution LB0890558 39488 10/23/08 10:29 TEST 0.721 546,800 Large 10-25

mX1 Large 10-25

mX1 NA

6 Pooled Pre-Wash Solution LB0890558 39488 10/23/08 10:29 TEST BDL 321,400 Small 10-25m X1 Small 10-25m X1 NA

X = Same Chromatographic Profiles as the Control.X1 = Same Chromatographic Profiles as the other Test Sample, No Control to Compare.2 = No peaks at 15.1 and 16.1. Those peaks are present in both “controls” and the other “test”.

BAXTER CONFIDENTIAL


3 = Peaks at 15.1 and 16.1 that were not present in the “controls” or the other “test”.m = minute peakNA = Not ApplicableCNBD = Could Not Be Determined due to No or Low Refractive Index Response.

Table 3 (Continued): Set 3 – LA Samples, Received 11-13-08 and 11-21-08

RLNumber


Media Lot Number


Total Protein(mg/mL)


RI SEC-UV at 280nm

SEC-UV at

220nm

LC-MS

4 Pooled Filtrate Sample after Post Wash LB0820580 39611 11/10/08

11:40

CONTROL 4.884 119,500 --- --- --- ---


11:40

CONTROL 4.708 128,000 --- --- --- ---


11:40

TEST 4.923 116,000 X X X X


11:40

TEST 4.979 118,900 X X X X


14:30

CONTROL 10.036 1,155,000* --- --- --- ---


14:30

CONTROL 10.312 29,460,000* --- --- --- ---


14:30

TEST 9.925 434,200,000* X X X X


14:30

TEST 9.944 49,930,000* X X X X


10:15

CONTROL 5.794 138,200 --- --- --- ---


10:15

CONTROL 5.271 140,200 --- --- --- ---


10:16

TEST 4.885 130,300 X X X X


10:16

TEST 5.452 136,000 X X X X


11:29

TEST 9.573 444,400 X2 X2 X2 X2

5 Filtrate Sample at the End of Filtration LB0820580 39611 11/10/08

11:08

CONTROL 4.921 125,300 --- --- --- ---


11:10

CONTROL 5.084 130,000 --- --- --- ---


11:08

TEST 4.818 119,900 X X X X


11:08

TEST 5.158 121,400 X X X X


14:05

CONTROL 12.401 37,360,000* --- --- --- ---


14:05

CONTROL 12.318 3,196,000* --- --- --- ---


14:05

TEST 11.862 274,800,000* X X X X


14:05

TEST 12.562 364,900,000* X X X X


10:00

CONTROL 5.623 141,600 --- --- --- ---

52 Filtrate Sample at the End of Filtration LB0820548 39602 10/28/0 10:0 CONTROL 5.682 137,700 --- --- --- ---

BAXTER CONFIDENTIAL


8 041 Filtrate Sample at the End of Filtration LB0820548 39488 10/28/0

810:03

TEST 5.358 132,200 X X X X


10:03

TEST 5.501 142,500 X X X X


11:05

TEST 11.213 10,160,000* X2 X2 X2 X2

53 11+ 111 EXTRACT BUFFER 705100323 --- 11/14/08

--- --- BDL CNBD Same as 54 25-35m 30m Same as 54

54 11+ 111 EXTRACT BUFFER 705100323 --- 11/14/08

--- --- BDL CNBD Same as 53 25-35m 30m Same as 53

55 40% POST WASH 702208318 --- 11/18/08

--- --- BDL CNBD Large 30-40m 10-15 m and 25-35m

30m Same as 56

56 BUFFER FOR SUSPENSION B (17%) POST WASH

702308099 --- 11/10/08

--- --- BDL CNBD Large 30-40m 25-35m 30m Same as 55

57 BUFFER FOR SUSPENSION B (17%) POST WASH – NO SAMPLE, CONTAINER BROKEN

702308099 --- 11/10/08

--- --- NA NT NT NT NT NT

X = Same Chromatographic Profiles as the Control.X1 = Same Chromatographic Profiles as the other Test Sample, No Control to Compare.X2 = No Control to Compare.m = minute peakNT = Not Tested

* = High molecular weights were obtained for Lot LB0890585. There was a significant light scattering response in the high molecular weight region for both the control and test samples. This response was reduced after filtration, however, the molecular weights were still high and the RI profiles molecular weight s for Lot LB0890585 were consistent for both the control and test samples.

BAXTER CONFIDENTIAL


The results in Table 3 show that the majority of samples showed no differences in the chromatographic profiles for SEC-RI, SEC-UV at 280nm, SEC-UV at 220nm or LC-MS. There were some minor absences or presences of the 15 and 16 minutes peaks that were seen in the “test” Starting Material (Suspension before CUNO Clarification). Since the starting materials had not passed through the CUNO resin, the “test” CUNO resin results had no impact on the production of Lots LB0820580, LB0890585 and LB0820548. There were some differences observed for the “test” Pooled Pre-Wash Solutions compared to its corresponding “control” samples. However, the majority of those differences showed an absence of peak for the “test” sample compared to its control. Since there was an absence of peak, no further testing was performed. For the “test” Pooled Pre-Wash solution samples for RL numbers 1 and 6, there was the presence of peaks observed between 10 and 25 minutes. There was some protein measured in RL sample 1, so the presence of those peaks was most likely due to protein contamination. The peaks were also observed in much smaller concentration for RL sample 6 even though no protein was measured. For both “test” samples, there was no corresponding “control” sample to compare. In addition, the molecular weight results and SEC profiles were consistent between the “test” and “control” samples indicating that the “test” CUNO resin had no impact on the products integrity and the differences observed from the “test” CUNO resin had no impact on the production of Lots LB0820580, LB0890585 and LB0820548.

A fourth set of samples was received on 12-11-08. This sample set were either buffer of WFI resin washes. Since these samples contained no protein, the BCA total protein method was not performed on these samples. The results for sample set 4 are summarized in Table 4.

BAXTER CONFIDENTIAL


Table 4: Set 4 – LA Samples, Received 12-11-08

Number Sample I.D. Suspension Lot Number

Media Lot Number Date Time Control or Test RefractiveIndex

UV at 220nm

UV at 280nm

LC-MS NMR GC-MS

27 Pre-Wash Solution NA 39611 12/05/08 11:46 CONTROL --- --- --- --- NA NA64 Pre-Wash Solution NA 39611 12/5/08 11:46 CONTROL --- --- --- 12.6 to 15.9m1 NA NA1 Pre-Wash Solution NA 39500 12/05/08 11:46 TEST X X X X NA NA

26 Pre-Wash Solution NA 39500 12/05/08 11:46 TEST X X X X NA NA61 Pre-Wash Solution NA 39611 11/25/08 15:48 CONTROL --- --- --- --- NA NA67 Pre-Wash Solution NA 39611 11/25/08 15:48 CONTROL --- --- --- --- NA NA68 Pre-Wash Solution NA 39500 11/25/08 15:48 TEST X X X X NA NA58 Pre-Wash Solution NA 39500 11/25/08 15:48 TEST X X X X NA NA10 Pre-Wash Solution NA 39611 12/09/08 09:33 CONTROL --- --- --- --- TBD TBD15 Pre-Wash Solution NA 39611 12/09/08 09:33 CONTROL --- --- --- --- TBD TBD20 Pre-Wash Solution NA 39500 12/09/08 09:33 TEST X X X Peak at 20.82 TBD TBD14 Pre-Wash Solution NA 39500 12/09/08 09:33 TEST X X X Peak at 20.82 TBD TBD17 Pre-Wash Solution NA 39602 12/08/08 15:00 CONTROL --- --- --- --- NA NA24 Pre-Wash Solution NA 39602 12/08/08 15:00 CONTROL --- --- --- --- NA NA4 Pre-Wash Solution NA 39488 12/08/08 15:00 TEST X X X X NA NA

18 Pre-Wash Solution NA 39488 12/08/08 15:00 TEST X X X X NA NA31 Pre-Wash Solution NA 39602 12/04/08 10:33 CONTROL --- --- --- --- NA NA46 Pre-Wash Solution NA 39602 12/04/08 10:33 CONTROL --- --- --- --- NA NA37 Pre-Wash Solution NA 39488 12/04/08 10:33 TEST X X X X NA NA36 Pre-Wash Solution NA 39488 12/04/08 10:33 TEST X X X X NA NA53 Pre-Wash Solution NA 39602 11/25/08 10:47 CONTROL --- --- --- --- NA NA54 Pre-Wash Solution NA 39602 11/25/08 10:47 CONTROL --- --- --- --- NA NA59 Pre-Wash Solution NA 39488 11/25/08 10:47 TEST X X X X NA NA74 Pre-Wash Solution NA 39488 11/25/08 10:47 TEST X X X X NA NA21 Pre-Wash Solution NA 39214 12/08/08 10:04 CONTROL --- --- --- --- NA NA3 Pre-Wash Solution NA 39214 12/08/08 10:04 CONTROL --- --- --- --- NA NA6 Pre-Wash Solution NA 39446 12/08/08 10:04 TEST X X X X NA NA7 Pre-Wash Solution NA 39446 12/08/08 10:04 TEST X X X X NA NA

38 Pre-Wash Solution NA 39214 12/03/08 11:23 CONTROL --- --- --- --- NA NA30 Pre-Wash Solution NA 39214 12/03/08 11:23 CONTROL --- --- --- --- NA NA34 Pre-Wash Solution NA 39446 12/03/08 11:23 TEST X X X X NA NA43 Pre-Wash Solution NA 39446 12/03/08 11:23 TEST X X X X NA NA52 Pre-Wash Solution NA 39214 11/24/08 15:05 CONTROL --- --- --- --- NA NA66 Pre-Wash Solution NA 39214 11/24/08 15:05 CONTROL --- --- --- --- NA NA56 Pre-Wash Solution NA 39446 11/24/08 15:10 TEST X X X X NA NA57 Pre-Wash Solution NA 39446 11/24/08 15:10 TEST Peak at ~ 12min X X X NA NA

1= Peaks in “control” from 12.6 to 15.9miutes but not seen in the other “control” or either “Test”.2 = Strong Peak at 20.8 minutes. Sample was submitted for NMR and GC-MS Anlaysis.NA = Not ApplicableTBD = To be Determined.

BAXTER CONFIDENTIAL


The results in Table 4 show that the majority of samples showed no differences in the chromatographic profiles for SEC-RI, SEC-UV at 280nm, SEC-UV at 220nm or LC-MS. The only sample that showed a difference was RL samples 20 and 14. These “test” samples along with their corresponding “control” were submitted for NMR and GC-MS analyses and will be reported in the final report. The only other observation was a peak observed in the SEC-RI profile at ~ 12 minutes for “test” RL sample 57. No differences were found for this sample when compared by SEC-UV at 220nm, SEC-UV at 280nm or LC-MS. Since the other control (RL sample 56) was consistent with its corresponding “controls” no further test was performed.

CONCLUSION

Based on the testing performed in this study, the “test” CUNO resin has no impact on product integrity during the production of Suspension B @ 17%, II+III Extract and Fr IV-4 @ 40% for albumin and IGIV from the LA facility. The few differences observed from the “test” CUNO resin when compared to its corresponding control can be attributed to ether protein contamination or higher alcohol content.

DOCUMENTATION

Original data and reports associated with this study will be archived following SOP 190501027, Multi-Divisional Archive Service Procedure.

References:

1. Feasibility Study of Resin Change for CUNO 70CP, by Trang Huynh, Bioscience. Test Protocol LB08EXP141. Project Number 36819555. September 26th, 2008.

Procedures:

1. Technology Resources Standard Procedure YY-005-004, Recording Original Data. Current Issue.

2. Technology Resources Standard Procedure YY-005-005, Writing Reports. Current Issue.

3. Multi-Divisional Archive Service Procedure Specification 190501027. Current Issue.

4. Technology Resources Standard Procedure PB-011-001. Use and Operation of High Performance Liquid Chromatography Systems. Current Issue.

5. Technology Resources Standard Procedure PA-011-005, Operation and Maintenance of the Bruker Avance III 600 AND Avance I 400 NMR Spectrometers. Current Issue.

6. Technology Resources Standard Procedure 1109105, Calibration, Operation and Maintenance of Mass Spectrometers and their Data Systems. Current Issue.

7. Technology Resources Standard Procedure 1096378, Operation and Calibration of the Digilab FTS 7000 Fourier Transform Infrared Spectrometer. Current Issue.

BAXTER CONFIDENTIAL

dtr 1, 45273, cuno, 2-26-09

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