Downloaded from on April 2, 2020 by guestMicrobiological Evaluation of Filters for Sterilizing Liquids, vol. 4. '1 Health Industry Manufacturing Association, Task Force on Filtration

OPS H. pseudoflava Filter paper 11/3/2009

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Filters with a 0.2 µm rating: Hydrogenophaga pseudoflava penetration 2

can be used to quantitatively assess impact of filtration parameters.* 3

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Lee, A. 1; J. McVey2 P. Faustino; S. Lute, 1; N. Sweeney3; V. Pawar2; 6

M. Khan4; K. Brorson1; D. Hussong2 7

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Office of Biotechnology Products1; Office of Pharmaceutical Science 9

New Drug Microbiology Staff2, Office Generic Drugs3, Office of Testing 10

and Research4; 10903 New Hampshire Ave.; Silver Spring MD 20903; 11

7520 Standish Place, Rockville, Maryland 20855. 12

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* This scientific contribution is intended to support regulatory 20

policy development. The views presented in this article have 21

not been adopted as regulatory policies of the Food and Drug 22

Administration at this time. Inclusion or exclusion of filter 23

brands or types in this study does not constitute an 24

endorsement or recommendation by the FDA or US 25

Government. 26

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01825-09 AEM Accepts, published online ahead of print on 4 December 2009

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Abstract 27

Filters rated 0.2 µm are used in laboratory and manufacturing settings 28

for diverse bacterial and particle removal applications from process 29

fluids, analytical test articles and gasses. Using Hydrogenophaga 30

pseudoflava, a diminutive bacteria with an unusual geometry (i.e. very 31

thin), we evaluated passage through 0.2 µm rated filters and the 32

impact of filtration process parameters and bacterial challenge density. 33

We show that consistent H. pseudoflava passage occurs through 0.2 34

µm rated filters. This is in contrast to an absence of significant 35

passage of nutritionally challenged bacteria that are of similar size 36

(i.e., hydrodynamic diameter), but dissimilar geometry. 37


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Introduction 38

39

The 0.2 µm filter class includes a large and diverse set of products 40

(22). They include air filters, particle reduction filters, filters used for 41

bioburden reduction, lab grade filters, and “sterilizing grade” filters 42

used in sterile dosage form manufacture. ASTM F 838-05, the 43

Brevundimonas diminuta challenge test, is a standard for the 44

“sterilizing grade” filters (4), a subset of the 0.2 µm rated filters. The 45

“0.2 µm” designation is applied to the larger and more diverse set of 46

products. This designation is based on physical measurements (e.g., 47

bubble point, the force necessary to extrude air through the capillary 48

network of wet filter) and mathematical extrapolations (5, 14, 29). 49

50

The current filter validation approach for parenteral pharmaceuticals 51

involves a demonstration of removal of 7 log10 cfu/cm2 of nutritionally 52

starved B. diminuta from bulk drug product liquids (4, 8, 11, 29). B. 53

diminuta can penetrate 0.2 µm rated filters, but only sporadically and 54

at low levels (12, 21). Larger bacteria (Listeria monocytogenes) have 55

been demonstrated to be able to penetrate 0.2 µm filters after long-56

term exposure (27). Recently, a species of small water-borne 57

bacteria, H. pseudoflava, have been shown to penetrate 0.2 µm rated 58

filters (31-36) to a greater extent than the above described bacteria. 59

None of these bacteria are actually physically smaller than 0.2 µm, 60

even H. pseudoflava (25, 37, 38). 61

62

Because H. pseudoflava penetrates 0.2 µm rated filters in a potentially 63

quantifiable manner, it can be used to study filtration efficiency. In 64

this report, we evaluate the impact of filtration process parameters 65

and bacterial challenge density on passage. We benchmark H. 66


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pseudoflava passage against nutritionally challenged bacteria which 67

are of similar size (i.e., hydrodynamic diameter), but dissimilar 68

geometry. 69

70

71

Materials and Methods 72

73

Bacteria. Hydrogenophaga (38) (formerly Pseudomonas) pseudoflava 74

(A.T.C.C. 700892); Brevundimonas (37) (also formerly Pseudomonas) 75

diminuta (A.T.C.C. 19146); Serratia marcescens (10) (A.T.C.C. 13880 76

& A.T.C.C. 8100) and Ralstonia pickettii (39) (A.T.C.C. 700590 & ATCC 77

49129) were purchased from American Type Culture Collection 78

(Manassas VA) or Fisher (Waltham MA). 79

80

Growth conditions. H. pseudoflava stock was prepared by seeding 81

with a 1:100 overnight culture and growth for 2 days in R2A media at 82

28oC with mixing at 100 RPM. B. diminuta was cultured 2 days in 83

Saline-Lactose Broth (SLB) at 30oC with mixing at 100 RPM after 84

seeding with a 1:100 TSB overnight culture . S. marcescens and R. 85

pickettii were cultured at 30oC in high purity water for 2 days with a 86

1:100 seed from a NB overnight culture. 87

88

Filters. Filters discs for bioprocessing & cell culturing from several 89

manufacturers were studied (Millipore Corp., Bedford, MA; Pall Corp., 90

Ft. Washington, NY; Whatman, Maidstone, UK; Sartorius, Goettingen, 91

Germany; Nalgene, Rochester, NY). These filter discs (0.2 µm rated) 92

were selected and purchased based on availability from lab supply 93

sources. Small disc filters (25-35 mm) were chosen to represent 94

multiple membrane chemistries (Table 1). 95


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96

Bubble point procedure. Filters were pre-wetted by flowing 150 mL 97

deionized water from a pressurized reservoir at 1 Bar, followed by a 5 98

minute equilibrium pause. The remaining water in the reservoir was 99

sent through the filter at 5-10 psi until the water flow stopped at the 100

filter surface. Pressure was increased gradually (~ 2 psi/min) until air 101

bubbles were seen passing from the filter outlets. A minimum of six 102

filters were tested to establish the water wet bubble point for each lot. 103

104

Growth Media and Chemicals. Glycerol, sucrose and NaCl were 105

purchased from Fisher Scientific (Waltham MA). Bacterial growth 106

media were purchased from Fisher. R2A was produced using ATCC 107

recipe 2258 with components purchased from Sigma (St Louis MO) 108

and Fisher. 109

110

Viscosity, pH, conductivity & osmolality Instruments. Challenge 111

solution viscosity was measured with Brookfield LVDV-III Ultra 112

Programmable Rheometer (Middleboro MA) with Enhanced UL Adapter 113

(1.0-2000 cP). The pH was measured with an Oakton pH6 Acorn 114

series pH meter (Singapore). The conductivity was measured with 115

Oakton Con 100 Series Conductivity Meter (Singapore). Osmolality 116

was measured with Wescor Vapro 5520 Vapor Pressure Osmometer 117

(Logan UT). 118

119

Model challenge fluids. H. pseudoflava was grown for 2 days and 120

then was mixed 1:1 with Minimal Media Davis (MMD), or with 60% 121

sucrose in MMD, or with 100% glycerol or with 1M NaCl in MMD. These 122

challenge solutions were mixed and allowed to equilibrate to room 123


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temperature for approximately 60 minutes prior to filtration 124

experiments. 125

126

Filter studies. Filter discs were challenged with bacterial loads 127

(ranging from 1 x 108 – 3 x 1010 cfu/cm2) from pressurized reservoirs 128

at a constant pressure of 2 bar. Challenge solutions were filtered into 129

sterile beakers inside a tissue culture laminar air flow hood. 130

Throughput was measured using precision balances. The instantaneous 131

liquid flow rate was calculated from the throughput readings and 132

filtering time measured by precision timers. Filtration was allowed to 133

proceed until the entire reservoir (200 mL) was filtered or the flux 134

decayed to as low as 0.1% of its initial water flux. Bacterial titers in 135

effluents and test solutions were measured by colony counting media 136

plates that had been overlaid with bacterial trapping membranes from 137

Millipore MicrofilTM filter cups (membrane/agar devices). Colony 138

morphology after 2-7 days incubation verified the correct species of 139

bacteria. A minimum of three filter discs were tested for each 140

challenge solution/ filter combination. 141

142

Log10 Reduction Value (LRV). LRV is calculated as the log10 (pre-143

filter bacterial titer ÷ post-filter bacterial titer). 144

145

Dynamic Light Scattering (DLS) Instrument. Light scattering was 146

performed with a Malvern Zetasizer nano S (Worcestershire, UK) with 147

an He-Ne laser (633nm) powered at 4mW. The instrument takes 12 148

or more consecutive measurements per sample, checking them for 149

reproducibility. The software (Dispersion Technology Software DTS 150

v5.02) includes an “expert advice” error checking function that allowed 151

elimination of readings that failed machine criteria. Bacterial test 152


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articles were 5 µm filtered prior to analysis. Each sample was read in 153

triplicate at one or more dilutions (technical replicates); results 154

passing machine criteria were averaged. Biological replicates were 155

read on different days and then averaged. 156

157

Scanning Electron Microscopy (SEM). SEM of bacteria was 158

performed by JFE Enterprises (Brookeville MD). Briefly, after the 159

specimen was fixed in 2.5% glutaraldehyde, it was then rinsed in 160

phosphate buffer. The free floating samples were positioned on a filter 161

before continuing. Biological samples were post-fixed in Osmium 162

Tetroxide for 1 hour and then washed in distilled H2O. They were then 163

dehydrated through a graded series of ethanol washes, critical point 164

dried, mounted on SEM stubs and coated with Au/Pd. A Hitachi 165

S-4700 FESEM instrument was used at the settings of an accelerating 166

voltage of 5.0KV, and a working distance of approximately 12mm. 167


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Results 168

Recently, H. pseudoflava has been shown to pass through 0.2 µm 169

rated filters with a variable LRV between 4-7 log10 (31-36). Because 170

H. pseudoflava passage was observed to be quantitative, we wanted to 171

see if it can be used to evaluate filtration efficiency under various 172

conditions. We first confirmed the observation that H. pseudoflava 173

passage occurred consistently across several 0.2 µm rated filter types 174

(Table 1). Under similar filtration conditions passage of three other 175

benchmarking bacteria (B. diminuta, S. marcesens, R. picketti) did not 176

occur. 177

178

Test fluid composition. 179

Extremes of process fluid composition (e.g. osmolarity, ionic strength, 180

and viscosity) can, in theory, impact bacterial passage. Previous 181

studies have shown that osmotic shock (after adjustment with sucrose 182

or NaCl) can result in either shrinkage or swelling of bacterial cells, 183

which presumably could impact filter passage (3, 6, 20). Impacts on 184

the membrane itself, such as charge shielding, are also in theory 185

possible. Discussions of the diversity of potential impact of these 186

parameters have been published (23, 24). However, it was noted 187

that retention of B. diminuta was robust even under extremes of these 188

parameters and the impact of parameters was discussed on a 189

theoretical level, in the absence of quantitative data actually assessing 190

impact. 191

192

To test these hypothesis with H. pseudoflava, model process fluids 193

were prepared to test extremes of osmolarity (30% sucrose, 1285 194

mmol/kg vs. MMD diluted culture, 99 mmol/kg); ionic strength (500 195


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mM NaCl, 54.4 mS/cm vs. MMD diluted culture, 9.1 mS/cm), and 196

viscosity (50% glycerol, 6.7 cP vs. MMD diluted culture, 1.1 cP). 197

198

Challenge studies of filters with various membrane chemistries by 199

high-density stock cultures of H. pseudoflava were performed with all 200

three challenge solutions as well as an MMD control (Fig. 1). For some 201

membranes, the passage did not appear to be impacted by extreme 202

process fluid physicochemical attributes; these membranes were 203

Aluminum Oxide (AlO3), Cellulose Acetate derivatives (CA’s), 204

Polyethersulfones (PES). For Nitrocellulose derivative (NC) 205

membranes, high levels of viscosity, osmolality and conductivity 206

increased H. pseudoflava retention. For Polysulfone (PS) membranes 207

50% glycerol increased bacterial retention. 208

209

Note the error bars (standard error of the mean) in our small scale 210

experiments, suggesting there can be variability in LRV between filter 211

runs. This may be due to (1) variation between small disc devices, (2) 212

inherent variability of H. pseudoflava passage or (3) effects from the 213

high load challenge conditions in our studies. Despite the variation, 214

the differences seen between process fluids for PS and NC membranes 215

are statistically significant (p < 0.05 in a t-test). 216

217

Bacterial challenge density. To evaluate the impact of challenge 218

density on filter performance, PES membranes from three 219

manufacturers were challenged with three levels of H. pseudoflava 220

(Table 2). High, medium and low challenges were prepared to be 221

about 1 log10 apart (undiluted bacterial culture, 1/10, 1/100 dilution). 222

For all three filter types, the lower challenge density leads to lower 223

LRV. There was some variability between vendors at the highest 224


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challenge density (range 4.5 log10 to 8.3 log10). SEM micrographs 225

taken of filters challenged at these high bacterial densities showed 226

some caking of bacteria on the membrane surface (data not shown). 227

In contrast, the retention levels at the lower challenge densities were 228

tighter between vendors (range 1.8 log10 to 3.6 log10), where the 229

lowest degree of retention (the highest passage of bacteria) occurred 230

in the filter type with the highest initial water flux. 231

232

These observations argue that filter type differences have less of an 233

impact on LRV than challenge density, possibly due to variable effects 234

of “caking”. This observation is unlikely to be an experimental artifact 235

given that (1) the same trend was observed for all 3 vendors, and (2) 236

the low LRV was not due to small experimental window (i.e., 237

H.pseudoflava was present in the filtrate even at lowest challenge 238

density). 239

240

Size and geometry of H. pseudoflava vs. three benchmarking bacteria. 241

To determine if shape differences (i.e., aspect ratio) can explain H. 242

pseudoflava passage vs. the other benchmarking bacteria (B. 243

diminuta, S. marcescens and R. pickettii), SEM micrographs were 244

taken from all four bacteria grown under low nutrient conditions (SLB, 245

R2A, H20; Fig. 2, Table 3). H. pseudoflava (Fig 2A) appears thinner 246

and longer than the other three bacteria, perhaps it is thin and flexible 247

enough to pass through the 0.2 µm filter pore network, unlike the 248

other three thicker bacteria (Fig, 2B, C and D). 249

250

We also measured the bacteria by Dynamic Light Scattering (DLS). 251

DLS measures Brownian motion of particles typically in the submicron 252

region, like bacteria, and relates this to the size, i.e., the larger the 253


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particle, the slower the Brownian motion (7). The size of a particle is 254

calculated from the translational diffusion coefficient by using the 255

Stokes- Einstein equation. 256

257

As can be seen, H. pseudoflava has approximately the same 258

hydrodynamic diameter as that of B. diminuta, R. pickettii, and S. 259

marcescens within the error of the assay (Table 3). The diameter that 260

is measured in DLS is a value that refers to how a particle diffuses 261

within a fluid (i.e., hydrodynamic diameter). This value will depend 262

not only on the size of the particle core, but also on the aspect ratio, 263

any surface structure, as well as the concentration and type of ions in 264

the medium. It should be noted that club-shaped bacteria are not 265

spheres, and can clump. Thus, sizes from DLS are approximations of 266

overall volume, not an exact diameter measurement. In this case, 267

long, thin bacteria can have a similar hydrodynamic diameter as short, 268

thick bacteria. 269


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Discussion 270

271

In this study we confirmed that H. pseudoflava passage occurred 272

consistently across 0.2 µm rated filters of several membrane types 273

from different vendors and with different matrix chemistries. 274

H. pseudoflava challenge testing results in quantitative result (LRV) vs. 275

qualitative result (+ or -) typical of a B. diminuta validation study (8, 276

29). 277

278

For a subset of the 0.2 µm rated membrane types studied, H. 279

pseudoflava passage was impacted by extremes in physicochemical 280

attributes (i.e., viscosity, osmolality and conductivity) of the model 281

process fluids. In theory, filtration or process fluid parameters that 282

modify the physicochemical attributes of the filter membrane or 283

bacteria can impact passage. For example, fluid osmolality is 284

predicted to shrink bacteria, increasing passage (23, 24). In other 285

publications, high viscosity has either been suggested to either 286

decrease retention by hampering Brownian motion-mediated 287

adsorptive effects (26) or by increasing processing time (23, 24). 288

Ionic strength has been suggested to decrease adsorptive effects by 289

shielding charge groups (24, 26), again potentially increasing passage. 290

Here, however, we demonstrate that the opposite predicted by theory 291

happened. While this observation cannot be ascribed to any specific 292

mechanism, one can speculate that our challenge solutions either 293

aggregate bacteria or have an impact on the filter membrane that 294

inhibits passage. The fact that we observed an impact of process fluid 295

on LRV for some but not other membrane types argues that it is most 296

likely that we were observing an effect on the filter, not the bacteria. 297

298


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Lower H. pseudoflava challenge densities in the model fluids correlate 299

with lower LRVs. This observation provides supportive evidence for 300

the theory that a caking effect, seen at high challenge densities, can 301

act as a “prefilter” to trap bacteria and inhibit passage. 302

303

We examined size, shape and 0.2 µm rated filter passage of three 304

other bacteria, B. diminuta, S. marcescens and R. pickettii as 305

benchmarked against H. pseudoflava. Only H. pseudoflava 306

experienced consistently passed though 0.2 µm rated filters. When 307

measured by DLS, all four bacteria were within 800-1200 nm in 308

hydrodynamic diameter, although SEM analysis revealed that they 309

possessed different shapes. The SEM results suggest that H. 310

pseudoflava is probably particularly prone to 0.2 µm rated filter 311

passage due to its geometry rather than its net volume; it is long and 312

thin relative to the other bacteria. Filters possess complex and 313

irregular matrix structures, not series of symmetrical, cylinder pores of 314

uniform sizes. Because of the “pore” shape, torturosity and 315

connectivity, and because of H. pseudoflava’s geometry, it may be 316

particularly prone to penetrate the passages of 0.2 µm rated filters. 317

318

In addition, the quantitative nature of H. pseudoflava passage is 319

particularly useful for process design and Quality by Design (QbD) 320

studies (13). Parameters such as processing time, transmembrane 321

pressure or fluid characteristics such as osmolality or conductivity can 322

be varied for assessment of their quantitative impact on H. 323

pseudoflava retention. 324

325

Arguments have been made that pharmaceutical facility flora are the 326

only relevant bacterial species to consider when designing filter 327


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validation studies (1, 2, 16). Also, there is no assumption by industry 328

or regulators that 0.2 µm rated membranes alone provide sterility 329

assurance in an absolute sense for injectable pharmaceuticals, rather 330

filtration is considered to be one part of a larger, multi-layer sterility 331

risk-mitigation strategy (8, 29). Thus, it is not warranted to conclude 332

that the observation of H. pseudoflava passage through 0.2 µm rated 333

filters should prompt a re-evaluation of filtration approaches for 334

injectable medicines (i.e., switching to 0.1 uM rated filters; (1, 15-335

19)). 336

337

The “0.2 µm” designation relies on an inverse relationship between the 338

largest pores in the membrane and the pressure needed to overcome 339

the capillary pressure in the pore network (5, 29). Based on the 340

bubble point pressure (P), a few assumptions described below, 341

parameters dependent on the filter and process fluid physico-chemical 342

attributes (e.g. liquid surface tension, γ; advancing contact angle of 343

liquid with respect to the pore wall, cos Θ), and a correction factor for 344

pore shape (k), the bubble point equation 345

346

P = (4kγ cos Θ) ÷ d 347

348

can be used to calculate d, the largest pores in the membrane (5, 14, 349

29). Pore sizes calculated from physical measurements depend on a 350

variety of assumptions, most notable of which is that the pores in a 351

typical filter membrane are simple circular capillaries extending from 352

one side of the membrane to the other. 353

354

However, almost all filters possess complex irregular matrix structures, 355

not a series of symmetrical, cylindrical pores of uniform size (14, 40) 356


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and capture particles by a variety of mechanisms (9). Because of this, 357

pore shape, torturosity and connectivity factors and aspect ratios 358

impact pore geometry, making the pore-size concept difficult to 359

interpret. Pore structure geometry varies widely between filter types 360

and brands, making an estimate of the k parameter difficult. In fact, 361

average pore sizes actually measured by scanning electron microscopy 362

(SEM) tend to be larger than pore size ratings (40). Studies with latex 363

beads have shown that 0.2 µm rated filters can allow passage of 0.5 364

µm beads under certain conditions (28). Further, the physico-365

chemical attributes that are parameters for the above equation (i.e., γ 366

and cos Θ) will vary with matrix chemistry, test fluid attributes and 367

filter type (14, 29, 30). 368

369

In light of the above described limitations of the 0.2 µm rating (i.e., 370

reliance on assumptions about pore geometry, influence of difficult to 371

measure physical factors like “wetting angle”) and the quantitative 372

nature of H. pseudoflava passage, a functional rating based on a set 373

level of H. pseudoflava retention may be a meaningful supplement to 374

the 0.2 µm designation. 375

376

It should be noted that these studies are preliminary and do not 377

constitute regulatory recommendations or policy. Current 378

recommendations and procedures for filter validation, including 379

challenge testing with B. diminuta should be followed as stipulated by 380

guidance. Filtration is one part of comprehensive sterility assurance 381

programs in place at pharmaceutical facilities. Further, these 382

observations do not imply that H. pseudoflava passage constitutes a 383

significant pharmaceutical safety issue, that H. pseudoflava is a 384

routine facility isolate or that the current filter validation methods and 385


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sterility assurance procedures are inadequate. It should also be noted 386

that in our studies model challenge solutions were chosen based on 387

simplicity, and not on actual process fluids. Further, the filters used in 388

this study were tested “as provided”; lab-grade disc devices rated as 389

0.2 µm, not pharmaceutical grade capsules. The membranes were 390

presumed to be representative of membranes in sterilizing grade 391

cartridges, despite potential differences in composite structure or QC 392

testing. However, none of the above listed limitations detract from the 393

central observation that H. pseudoflava challenges result in a 394

quantitative result (LRV). 395

396

Conclusions 397

In conclusion, we find H. pseudoflava passage occurred consistently 398

across filters of several 0.2 µm membrane types from different 399

vendors and with different matrix chemistries. Factors that appear to 400

impact filter passage included: filter and process fluid physicochemical 401

attributes, bacterial load level and bacterial geometry. 402

403

Acknowledgments 404

This project was supported in part by (1) a Regulatory Science and 405

Review Enhancement program grant from CDER/FDA and (2) the 406

Research Participation Program at the Center for Drug Evaluation and 407

Research administered by the Oak Ridge Institute for Science and 408

Education through an interagency agreement between the U.S. 409

Department of Energy and the U.S. Food and Drug Administration. 410

411

We acknowledge Agnes Nguyen (CDER/FDA) for instruction in 412

operating the viscometer and osmometer. We thank Hans G. 413

Schroeder for stimulating discussions concerning 0.2 µm filtration. We 414


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also acknowledge the following individuals for careful peer of this 415

manuscript Keith Webber, Dennis Guilfoyle. 416

417

418

Figure Legends 419

420

Figure 1. Results of high H. pseudoflava challenge tests with three 421

model process fluids. Shown are polyethersulfone (n = 3), cellulose 422

acetate derivatives from two manufacturers (n =3), aluminum oxide (n 423

= 3) nitrocellulose derivative (n =3), polysulfone (n =3). Replicate 424

challenge tests were generally performed with independent cultures on 425

different days. Bars represent standard error of the mean. 426

427

Figure 4. SEM (20,000 x magnification) of bacteria grown under low 428

nutrient conditions. A) H. pseudoflava cultured 2 days in R2a. B) B. 429

diminuta cultured 2 days in SLB. C) R. pickettii cultured 2 days in H2O. 430

D) S. marcescens cultured 2 days in H2O. 431

432


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554


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Table 1. Log10 reduction value for 0.2 µm filtration of H. pseudoflava and three

benchmarking bacteria.

LRV (log10) Chemical Derivativea Vendor

H. pseudoflava Benchmarking bacteriab

Polyethersulfone (PES) A 4.6 ± 0.4 >9

B 6.7 ± 0.4 >9

C 4.1 ± 0.4 >9

Cellulose Acetate (CA) A 3.9 ± 0.9 >9

E 4.6 ± 0.4 >9

Polyvinylidene fluoride

(PVDF)

B 6.8 ± 2.3 >9

Cc 7.5 ± 0.3 >9

Nitrocellulose (NC) B 5.5 ± 1.2 >9

Nylon C 5.6 ± 0.3 >9

Cc 6.5 ± 1.1 >9

Polysulfone (PS) C 6.9 ± 0.9 >9

Aluminum Oxide (AlO3) D 5.2 ± 0.5 >9

a. All filters were lab grade intended for tissue culture (TC): “lab-grade

filtration (sterilizing for tissue culture)”, ”Small volume liquid

sterilization, Sterile laboratory filtration devices”, “lab, sterilizing”,

“Sterilizing filter unit, also for TC” “cold sterilization, sterile filtration”,

“Small volume liquid sterilization, Sterile laboratory filtration devices”,

etc.

b. Data represents an average LRV from 3-8 filtration runs.

c. Benchmarking bacteria were B. diminuta, S. marcescens, and R.

picketti grown two days under low nutrient conditions (B. diminuta,

saline lactose broth; S. marcescens and R. picketti, distilled water).


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Generally, complete clearance was observed (LRV >9 log10). In a

minority of cases, sporadic, low level breakthrough (<1 cfu/10 ml)

occurred in minority of replicates.

d. Stated by manufacturer to be a positively charged membrane.


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Table 2. H. pseudoflava passage through PES 0.2 µm membranes at three challenge levels.

PES

Type

Mean

Bubble

Point (PSI)

Mean Water

Fluxa

(mL/min/cm2)

Test Undilutedb 1:10 Dilution 1:100 Dilution

Challenge log10(cfu)/cm2 10.0 to 10.4

9.3 to 9.7

8.3 to 8.7

A 46.1 ± 0.8 75.4 ± 3.4 LRV

c (log10) 4.5 ± 0.3 2.3 ± 0.1 1.8 ± 0.3

Challenge log10(cfu)/cm2 9.7 to 9.8

9.1 to 9.2 8.0 to 8.2

B 73.4 ± 0.2 38.1 ± 1.4 LRV

c (log10) 8.3 ± 1.5 5.3 ± 0.1 3.5 ± 0.2

Challenge log10(cfu)/cm2 9.2 to 9.8 8.9 to 9.6 8.1 to 8.8

C 49.4 ± 0.7 49.4 ± 1.4 LRV

c (log10) 5.3 ± 0.8 3.8 ± 0.8 3.6 ± 0.2

a. Pre-filtration water flux at 15 psi.

b. When H. pseudoflava was filtered undiluted (~109 cfu/mL) the volumetric throughput was limited by filter blockage.

c. Instantaneous LRV after extended throughput.


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Table 3. Summary of bacterial size information.

Bergey’s Manuala (µm) SEM Measurementc DLS Bacteria

Width Length Width Length Hydrodynamic diameterd

H. pseudoflava 0.3-0.6 0.6-5.5b 0.24 ± 0.01 2.48 ± 1.04 994 ± 193

B. diminuta 0.4-0.5 1-2 0.41 ± 0.08 1.03 ± 0.58 780 ± 89

S. marcescens 0.5-0.8 0.9-2.0 0.51 ± 0.05 1.03 ± 0.30 976 ± 67

R. pickettii 0.5-0.6 1.5-3.0 0.39 ± 0.03 0.89 ± 0.23 827 ± 85

a. Sections in Bergey’s Manual are (10, 37-39)

b. H. pseudoflava is 0.25 ± 0.03 x 1.65 ± 0.35 µm and B. diminuta is 0.31 ± 0.03 x 0.88 ± 0.19 µm

according to Sundaram (31-36).

c. Averages and standard deviations of 45 to 105 different bacteria measured in the SEM micrographs

in Fig. 2.

d. Measured on 2-7 independent samples on different days. Each measurement represents an average

of 3 or more independent measurements of each sample.


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